MANUAL OF LABORATORY EXERCISES
BIOL518: BIOTECHNOLOGY SKILLS LABORATORY I
CAL STATE LA
DR. HOWARD XU
Acknowledgements: I thank many of the former and current students in my research lab who
have contributed in compiling the laboratory exercises: Stephanie Tan, Lilian Real, Qiyu Huang,
Chris Lam, Tan Truong, Irving Philips and Isba Silva. I am grateful to Cal State LA’s Office of
Graduate Studies and Research and the College of Natural and Social Sciences for funding the
development and operation of this intensive laboratory course.
Table of Contents
Course Introduction
Housekeeping Rules
General Lab Equipment
Review of Basic Lab Techniques
Exercise 1. Micropipeting Basics (D1 and D2)
Exercise 2. Bacterial Transfer and Isolation Techniques (D1, D2 and D3)
Stage 1. Bioinformatics
Exercise 3. Target selection and target gene analyses (D1 and D2)
Exercise 4. Primer design (D2 and D3)
Stage 2. Molecular Cloning
Exercise 5. Ligation-Independent Cloning (D2 and continued W2)
Exercise 6. Bacterial Chromosome DNA miniprep (D1, D2 and D3)
Stage 2. Molecular Cloning (continued)
Exercise 7. Polymerase Chain Reaction (D1 and D2 scale-up)
Exercise 8. Agarose Gel Electrophoresis (D2)
Exercise 9. PCR Purification (D3)
Exercise 10. Transformation (D3, D4 volunteers ; W3-D2 transform DL21)
WEEK 1
Stage 2. Molecular Cloning (continued)
Exercise 11. Plasmid Miniprep (D0 volunteers, D1)
Exercise 12. Restriction Digest Analysis (D2, D3 streak/pure)
WEEK 3
Stage 3. Biochemistry
Exercise 13. Induction of Target Protein Over-expression
Exercise 14. Polyacrylamide Gel Electrophoresis
Exercise 15. Protein Purification (D3 on their own)
WEEK 4
Stage 3. Biochemistry (continued)
Exercise 15. Protein Purification
WEEK 5
Stage 4. Assay Development
Exercise 16. Assay Development
WEEK 6
Stage 5. High Throughput Screening
Exercise 17. Screening chemical libraries for enzyme inhibitors using HTS
robotics
WEEK 7
2
WEEK 2
Stage 5. High Throughput Screening (continued)
Exercise 17. Screening chemical libraries for enzyme inhibitors using HTS
robotics
Stage 6. Chemoinformatics/SAR
Exercise 18. Data analysis/chemoinformatics
Exercise 19. Hit-picking and confirmation assay
Exercise 20. Susceptibility testing (MIC)
Stage 7. Communication Module (Team Summary Report and Team Presentation)
Group work to prepare for Team Report and for Team Presentation
(ASC-126 is available for use; each group is free to schedule time and location to
meet and work on report and presentation)
WEEK 8
Stage 7. Communication
Team report due and team presentations
FINAL WK
Appendix.
3
WEEK 9
WEEK 10
Course Introduction
Welcome to the lab! In this course, you will learn the principles and practices of drug discovery using
antibiotic drug discovery as a model process. Through this course, you will understand the
multidisciplinary and project team nature of drug discovery process. You will learn and familiar with
many skills and techniques used in the biotechnology industry.
Objectives of the PABS PSM Skills Application Course:
•
Learn to work in a multidisciplinary team environment;
•
Become familiar with principles and practices of multiple disciplines beyond one’s own field of
expertise and training;
•
Apply the principles of chemical genomics to drug discovery;
•
Apply laboratory robotics to the drug discovery process;
•
Gain confidence in leading a stage of the drug discovery process as a task leader.
•
Improve oral and written communication skills.
Students will be assigned into groups that reflect the multidisciplinary nature of most project teams in the
biotechnology and pharmaceutical industries. An ideal team would have students from the following
undergraduate majors: computer science/applied math, molecular biology, biochemistry, engineering, and
chemistry. Within your teams, you should elect a project manager that will be responsible for keeping the
pace of the experiments on time and to make sure all deadlines are accomplished without delay. Each
stage of the project will have a major emphasis on a particular discipline (such as molecular cloning,
biochemistry, etc.) and teams should select a task leader for each stage based on a student’s area of
primary expertise.
Each team will be required to select its own drug target gene to clone and will follow parallel phases of a
drug discovery project to the end of identifying, confirming hits, and determining spectrum of activity of
the hits.
Note: The techniques in this book stress cleanliness and sterilization as this is a critical part in being a
microbiologist or scientist. There are bacteria everywhere and can easily have an adverse effect on your
research or your personal self! They can grow in a multitude of various conditions; they exist on you and
in you as normal flora. Some bacteria can be opportunistic and take advantage of you when your body is
weak. Although the ones we work with are specifically designed for our experimental purposes, it is
imperative that you still treat them as dangerous pathogens that can potentially infect you.
Anyway, good luck to everyone!
4
Housekeeping Rules
1. At the beginning and end of each lab period, spray the lab bench top down with general
disinfectant (4% amphyl) or 10% bleach solution. Let stand for 1 minute before wiping down
with a paper towel.
2. Wash your hands thoroughly before and after each lab period.
3. Always wear protective gloves when handling reagents.
4. Always wear proper clothing in the lab: long pants and closed toe shoes. Lab coats must be worn
at all times.
5. Long hair must be tied back.
6. No eating, drinking, or smoking in the laboratory. Avoid placing things in your mouth (fingers,
pen tops, etc…) and then laying them on your bench top.
7. Do not handle contact lens or do your makeup in the laboratory.
8. No extraneous books, folders, purses, etc. on the bench. Store them under the bench or on
counters along the wall. Only the necessary equipment, this lab manual, and your lab notebook
should be on your bench top.
9. Please REPORT ALL SPILLS AND ACCIDENTS IMMEDIATELY. Do not try to clean it up
without supervision of lab instructor.
10. Know the location of the fire extinguisher, eyewash, and safety shower.
11. Do not leave cultures in lockers unless specified.
12. Treat all organisms as pathogens.
13. Always use aseptic techniques (see General Lab Techniques).
14. Clean up after yourself. Don’t take community reagent bottles to your bench, and place all items
back into their respective location.
15. Discard your waste appropriately. Treat everything as biological hazards.
a. No liquid, unless specified, is to be discarded in the sinks.
b. Petri dishes and disposable tubes should be discarded in autoclave bags in coffins.
c. Plastic pipette tips and cotton swabs go into open plastic containers on your bench top.
d. Calibrated disposable pipettes can be discarded into paper “Pipet Keeper Containers” on
your bench top.
e. See your instructor if you are unsure about where to place waste from your
experiment.
16. Identify everything with your NAME, TYPES OF MEDIA/EXPERIMENT, ORGANISM,
DATE, and LAB SECTION.
a. Label your plates on the bottom – plates are incubated upside down to reduce potential
contamination and water condensation on the agar surface.
Tubes and plates may have to be refrigerated and may be easily shuffled around, and could get lost among
the confusion.
5
General Lab Equipment
Erlenmeyer flask
Graduated
cylinder
Inoculating
loop
Micropipette (variable volume)
Eppendorf or
microcentrifuge
tube
Petri dish
Helpful
Tip#1:
Beaker
PCR tube
Materials you should always bring
to class:
- lab manual
- lab notebook
- fine tipped permanent marker
- pen (always write in ink,
preferably black)
Materials you should leave in your
locker:
- lab coat
- a box of gloves
- pair of goggles
Gloves
Lab coat
6
EXERCISE 1. Micropipeting Basics
OBJECTIVE


Familiar with metric unit measurements and their conversions.
Able to accurately pipet different microliter volumes using a micropipet.
INTRODUCTION
Over the past several decades, advances in biotechnology have influenced many changes in experimental
techniques and methods including the volume of reagents and biological samples used. Depending on the
procedure being performed, biotechnology experiments can utilize a variety of biological samples and
reagents, ranging from several hundreds of liters to very small microliter (l) volumes.
Pipeting is a critically important technique in life science experiments to ensure accurate
experimental results. In typical biotechnology experiments, biologicals and reagents such as DNA,
enzymes, and buffers are transferred by pipeting into small microcentrifuge tubes that serve as reaction
vessels. For these types of reactions, microliter volumes are typically used. There are 1,000 microliters
in a milliliter of a solution. To put it in perspective, a 50 microliter sample is approximately equal to a
single raindrop. A raindrop-sized sample is relatively large when compared to experimental samples that
often are 10 to 50 microliters in volume.
To measure microliter volumes, a special instrument called a micropipet is used. The variable
volume micropipet is the preferred instrument for delivering accurate, reproducible volumes of sample
(Figure 11.1). These instruments are manufactured to deliver samples in various ranges (e.g., 0.5-10 l,
2-20 l, 20-200 l, 200-1000 l, etc.) and usually can be adjusted in one microliter increments. Typically
these instruments have an ejector button for releasing the tip after sample delivery. Variable volume
micropipets can also be multi-channeled, designed to uniformly deliver several samples at the same time.
However, for this experiment, only one sample will be delivered at a time.
Figure 1.2
7
1.
2.
3.
4.
Set the micropipet to the appropriate volume and place a clean tip on the micropipetor. Press the
top button down to the first stop and hold it in place while placing the tip into the sample fluid.
Once the tip is immersed in the sample, release the button slowly to draw the sample into the tip.
Deliver the sample by pressing the button to the first stop – then empty the entire contents of the
tip by pressing to the second stop.
Press the tip ejector to discard the tip. Obtain a new, clean tip for the next sample.
Figure 1.2 Sample delivery with variable automatic micropipets
MATERIALS
Per group of 5 students (4 groups for the entire class):
 p20 micropipet
 p200 micropipet
 Pipet tips
Per group of 5 students:
 Red reagent
 Blue reagent
 Yellow reagent
 Glycerol
 Alcohol
 Buffer
 7 microcentrifuge tubes
PROCEDURE
Activity One: Volumetric Applications of the Metric System
The metric system is used in micropipeting. The milliliter (ml) and microliter (l) are two very useful
units of measure in molecular biology. Milli means one-thousandth and micro means one-millionth.
8
There are 1,000 (103) milliliters in one liter. There are 1,000 (103) microliters in a milliliter and 1,000,000
(106) microliters in one liter. In scientific notation 1 microliter = 10-6 liter. In decimals 1 microliter =
0.000001 liters.
1.
Perform the following conversions:
In decimals
1 ml = _____________ liter
1 liter = _____________ ml
1ml = _____________ l
1 l = _____________ ml
10 l = _____________ ml
20 l = _____________ ml
50 l = _____________ ml
100 l = _____________ ml
In scientific notation
1ml = _____________ liter
1 liter = _____________ ml
1 ml = _____________ l
1 l = _____________ ml
10 l = _____________ ml
20 l = _____________ ml
50 l = _____________ ml
100 l = _____________ ml
2.
How many times greater is a ml than a l? ________
3.
How many times greater is a liter than a ml?
4.
How many times greater is a liter than a l?
Activity Two: Micropipeting Using a Variable Volume micropipet
1.
In the activity that follows, you will use a variable volume micropipet to prepare (mix) seven
different dye mixtures in microcentrifuge tubes.
2.
Label 7 microcentrifuge tubes with your initials and the numbers 1 to 7. Refer to Table 11.1
to prepare the 7 dye solutions.
Table 1.1
TUBE
RED (l)
BLUE
(l)
1
2
3
4
5
6
7
5
5
13
6
10
15
10
-
YELLOW GLYCEROL ALCOHOL BUFFER TOTAL
VOLUME
(l)
(l)
(l)
(l)
(l)
10
10
4
10
10
15
REFERENCES
(Modified from Micr300 Laboratory Manual, CSULA)
9
10
10
-
40
15
25
25
22
25
20
45
45
45
45
45
45
45
REPORT AND QUESTIONS (Exercise 1)
Your Name: _____________________
Answer the following questions.
1.
How does the pipeting exercise help you understand the importance of accurate pipeting
using microliter volumes?
2.
Why did you practice pipeting samples of various viscosities?
3.
Describe a good technique for withdrawing samples using a variable volume micropipet.
4.
When using the micropipets in the lab, what volume is being delivered when the following
digits appear in the window of a P20 micropipet? A P200 micropipet?
0
8
5
0
10
Review of Concentrations
Concentrations: are ratios of an amount of a chemical to a solvent in defined volume.
Percent solutions:
There are various types, but the most common is % w/v. % w/v means percent
weight to volume, and has units of g / 100 mL.
Molarity:
This type of concentration refers to the number of moles of solute in a total
volume of 1L. Moles are calculated by dividing grams of chemical by the formula weight
of that chemical (g/mol) and dividing the volume (L) of the solution.
Times (X):
Occasionally, some solutions are made as stock solutions, but they are usually
stored at a higher concentration than necessary by n times over. These require dilution
down to 1X. This is done by diluting 1 part of the nX solution with n-1 parts of the
diluent.
Helpful Tip:
Always watch your units!
Make sure they cancel out or come out on the correct side.
Don’t forget to take into account derivatives such as mM, µM, or even nM.
11
EXERCISE 2. Bacterial Transfer and Isolation Techniques
OBJECTIVE




Carry out the techniques for aseptic removal and transfer of microorganisms into liquid and solid
media including from broth-to-broth, broth-to-agar, agar-to-agar, agar-to-broth media.
Correctly sterilize inoculating instruments in the flame of the Bunsen burner.
Use disposable plastic inoculating loops
Isolate bacteria in pure culture from a mixed culture.
INTRODUCTION
Microorganisms are transferred from one medium to another by subculturing. This technique is of basic
importance and is used routinely in preparing and maintaining stock cultures, as well as in
microbiological test procedures. Microorganisms are always present in the air and on laboratory surfaces,
benches, equipment and human skins. They can serve as a source of external contamination and thus
interfere with experimental results unless proper techniques are used during subculturing. Aseptic
technique refers to the measures taken to prevent external contamination. Described below are essential
steps that you must follow for aseptic transfer of microorganisms.
1. An inoculating loop (or needle) must always be sterilized by holding it in the hottest portion
of the Bunsen burner flame, the inner blue cone, until the entire wire is glowing red. Then the upper
portion of the handle is rapidly passed through the flame. Once flamed, the loop is never put down but is
held in the hand and allowed to cool for 10 to 20 seconds. Although a microbiologist should be trained
and experienced in multiple types of culture transfer (including agar slants), for the purpose of this course,
we only require practicing and grasping techniques involving broth media (in tubes or flasks) and agar
media plates. The stock culture tube and the tube to be inoculated are held in the palm of the other hand
and secured with the thumb. The two tubes are then separated to form a “V” in the hand.
2. The tubes are uncapped by grasping the first cap with the little finger and the second cap with
the next finger and lifting the closures upward. Once removed, these caps must never be placed on the
laboratory bench because doing so would compromise the sterile procedure. Keep them in the hand that
holds the sterile inoculating loop while pointing the inner aspects of the caps away from the palm of the
hand. Following removal of the closures, the necks of the tubes are briefly passed through the flame (not
for sterile plastic tubes) and the sterile transfer instrument is further cooled by touching it to the sterile
inside wall of the culture tube (but not for plastic tubes) before removing a small sample of inoculum.
3. Depending on the culture medium, a loop or needle is used for removal of the inoculum. Loops
are commonly used to obtain a sample from a broth culture. Either instrument can be used to obtain the
inoculum from an agar culture by carefully touching the surface of the solid medium in an area exhibiting
growth so as not to gouge into the agar. A straight needle is always used when transferring
microorganisms to an agar deep tube from both solid and liquid cultures.
4. The loop or needle with the microorganism is inserted into the subculture tube. In the case of a
broth medium, the loop or needle is shaken slightly to dislodge the organisms; with an agar slant medium,
it is drawn lightly over the hardened surface in a straight or zigzag line. For inoculation of an agar deep
tube, a straight needle is inserted to the bottom of the tube in a straight line and rapidly withdrawn along
the line of insertion. This is a stab inoculation. For most of our applications, agar media plates will be
used on which a straight or zigzag line is draw with the loop.
5. Following inoculation, the loop or needle is removed, the necks of the tubes are re-flamed
(only for glass tubes), and the caps are replaced on the same tube from which they were removed.
6. The needle or loop is again flamed to destroy remaining organisms.
12
Pure Culture Preparation
In nature, microbial populations do not segregate themselves by species but exist with a mixture of many
other cell types. In the laboratory these populations can and must be separated into pure cultures. Pure
cultures contain only one type of organism in large amounts and are suitable for the study of their
cultural, morphological, and biochemical properties.
In this experiment, you will first use a technique designed to produce isolated or discrete
colonies. Colonies are individual, macroscopically visible masses of microbial growth on a solid medium
surface, each representing the multiplication of a single organism. Once you have obtained isolated
colonies, you will make an aseptic transfer onto nutrient agar plates for the isolation of pure cultures.
The techniques commonly used for isolation of discrete colonies initially require that the number
of organisms in the inoculum be reduced. The resulting diminution of the population size ensures that,
following inoculation, individual cells will be spaced sufficiently far apart on the surface of the agar
medium to allow the formation of distinct colonies and separation of the different species present. The
four-quadrant isolation method is a rapid qualitative isolation technique that can be used to accomplish
this necessary dilution by essentially spreading a loopful of culture over the surface of an agar plate. Refer
to Figure 1.1, which schematically illustrates this procedure.
1. Label the agar side of a sterile plate with the Roman numerals (I, II, III and IV) to designate the
four quadrants.
2.Using aseptic techniques place a loopful of culture on the agar surface in quadrant I. Flame the
loop, and cool it by touching an unused part of the agar surface close to the periphery of the plate, and
then drag it rapidly several times across the surface of Quadrant I.
3. Re-flame and cool the loop, and turn the Petri dish 90 . Then touch the loop to a corner of the
culture in quadrant I and drag it several times across the agar in quadrant II. The loop should never enter
Quadrant I again.
4. Re-flame and cool the loop and again turn the dish 90°. Streak Quadrant III in the same manner
as Quadrant II.
5. Without re-flaming the loop, again turn the dish 90° and then drag the culture from a corner of
Quadrant III across Quadrant IV, using a wider streak. Do not let the loop touch any of the previously
streaked areas. The flaming of the loop at the points indicated is to effect the dilution of the culture so that
fewer organisms are streaked in each area, resulting in the final desired separation.
6. Once discrete, well-separated colonies develop on the surface of a four quadrant streak plate,
each discrete colony may be picked up with a sterile loop or needle and transferred to separate nutrient
medium (broth or agar). Each of these new slant or plate cultures represents the growth of a single
bacterial species and is designated as a pure or stock culture, which can be will confirm by a Gram stain.
13
I. Heavy confluent growth
Flame loop
II. Heavy growth
IV. Isolated colonies
Flame loop
III. Light growth
Figure 1.1 Four Quadrant Isolation Streak Plate
MATERIALS
Subculturing



24-hour nutrient broth and nutrient agar plate cultures of Escherichia coli [4 sets (3 ml/tube) for 4
groups]
Per student: two nutrient broths, two nutrient agar plates. [40 tubes of nutrient broth; 40 nutrient
agar plates]
Bunsen burner, inoculating loop, test tube rack and magic marker. [one set per group]
Pure Culture Preparation
FIRST PERIOD




24-hour nutrient broth cultures E. coli and Staphylococcus epidermidis (to be mixed 1:1 ratio by
instructional support technician; 3 ml tube/group for 4 groups)
Per student: one nutrient agar plate. [20 nutrient agar plates].
Per group: one LB agar plate [4 LB agar plates for lab]
Bunsen burner, inoculating loop, test tube rack and magic marker.
SECOND PERIOD



Four quadrant streak plate from FIRST PERIOD.
Per student: two nutrient agar plates. [40 nutrient agar plates]
Bunsen burner, inoculating loop, test tube rack and magic marker.
THIRD PERIOD



Subculture from SECOND PERIOD.
Per student: two nutrient agar plates. [40 nutrient agar plates]
Bunsen burner, inoculating loop, test tube rack and glassware marker.
PROCEDURE
14
Subculturing
1. Label all tubes of sterile media with the name of the microorganism, date and your initials.
2. Following the procedure outlined and illustrated above, perform the following transfers:
a. E. coli broth culture to nutrient broth, and a nutrient agar plate.
b. E. coli agar plate culture to a nutrient broth, and a nutrient agar plate.
3. Incubate all cultures at 37°C for 24 hours.
4. (the next period) Examine the growth or lack of growth (indicating failed transfer)
Pure Culture Preparation
FIRST PERIOD
1. Label a nutrient agar plate on the side where the agar is with the name of the microorganisms,
date, your initials and the Roman numerals (I, II, III and IV) to designate the four quadrants.
2. Briefly shake the tube containing the mixed culture without spilling any liquid.
3. Aseptically transfer a loopful of the bacterial broth culture onto a corner of the nutrient agar.
4. Following the procedure outlined and illustrated above, perform a four-quadrant isolation streak.
5. Incubate the plate for 24 hours at 37C.
SECOND PERIOD
1. Aseptically transfer, from visibly discrete colonies of your four quadrant isolation plate, either E.
coli or S. epidermidis, to the appropriately labeled agar plates.
2. Incubate the cultures for 24 hours at 37C.
3. (per group) Transfer 3 ml of sterile LB broth into a Falcon tube aseptically for
THIRD PERIOD
1. Examine the culture morphology of your subcultures.
2. For each of the two subcultures, following aseptic techniques, repeat streaking one fresh nutrient
agar plate.
3. Incubate the cultures for over the weekend at room temperature.
REFERENCES
(Modified from Micr300 Laboratory Manual, CSULA)
15
REPORT AND QUESTIONS (Exercise 2)
Your Name: _____________________
A. Report
Draw your streaked plates:
E. coli streaking:
Colony/cell biomass distribution
and appearance
S. epidermidis streaking:
Colony/cell biomass distribution
and appearance
Mixed culture streaking:
Colony/cell biomass distribution
and appearance
Color:
Color:
Color:
Pigmentation:
Pigmentation:
Pigmentation:
Surface characteristics
(smooth?):
Surface characteristics
(smooth?):
Surface characteristics
(smooth?):
B. Please answer the questions:
1. Explain why the following steps are essential during subculturing:
a)
Flaming the loop or needle prior to and after each inoculation.
b)
Cooling the loop or needle prior to obtaining the microorganism.
c)
Holding the test tube caps in the hand as opposed placing them on the bench top.
2. How could you explain if a four-quadrant streak plate culture shows more growth in quadrant
IV than in quadrant III?
16
EXERCISE 3. Target Selection and Bioinformatics Analysis
OBJECTIVE


Understand the concept of therapeutic targets
Become familiar with criteria of target selection
INTRODUCTION
For thousands of years, peoples all over the world have established, via trial and error, particular ways of
treating ailments of various kinds. For example, Chinese have used extracts of mixed herbs and/or animal
parts for thousands of years to treat diseases. These treatments were the original therapeutics. Modern
chemotherapeutics began with Paul Ehrlich’s Salvarsan in early 1900’s for treating syphilis. This was
followed by use of sulfonamides and penicillin. Chemotherapeutics can be used to treat many types of
diseases such as cancer, heart diseases and infectious diseases. For the purpose of this lab, we concentrate
on antibiotics as therapeutic agents to treat bacterial infections.
In the early days of antibiotics, antibiotic drugs were approved for clinical use simply by how
well they worked by killing bacterial cells but not animals or humans. With advancement of bacterial
genetics and biochemistry, how these drugs act (mechanisms of action) were gradually elucidated with
genetic and biochemical experiments. Usually, antibiotics were found to inhibit the activities of essential
enzymes or proteins in bacterial cells. In 1995, the completion of genome sequence of the first cellular
organism (Mycoplasma genitaliam) ushered in the genomic era, leading to the sequencing of human
genome in 2003. Now, hundreds of microbial genomes have been sequenced. With the advance of
genomics, drug companies must fulfill the FDA requirement of the nature of drug targets and how they
work, among other requirements such minimal side effects, efficacy, metabolism and absorption, etc.
Currently available antibiotics all act on essential enzymes or pathways in bacteria. However, so
far less than 40 of the estimated 200 essential proteins have been targeted by currently available
antibiotics. Consequently, the reminder of the essential protein landscape could be potential targets of
novel antibiotics. For any company interested in developing an antibiotic drug, target selection is a critical
process that requires extensive analysis and experimentation. The most desirable antibiotic should be
effective in killing or inhibiting growth of as many types of bacteria as possible. In general, an antibiotic
effective against at least several species of both Gram positive and Gram negative bacteria is termed
broad spectrum antibiotic. In contrast, a narrow spectrum antibiotic is only effective against specific
species or genera of bacteria, usually within either Gram positive or Gram negative bacteria. If a potential
target is absent in many of the key pathogens or exists but is not well conserved among key pathogens
(which make it unlikely that an inhibitor would be equally effective against), Consequently, a drug target
should be fit the following criteria: (a) essential; (b) conserved among several key pathogens of both
Gram positive and negative bacteria; (c) novel (not similar to targets of any present drugs) and (d) cidal.
Bioinformatics is a field that combines biology and information technology. A staggering amount
of information gained through sequencing projects has been stored in databases that can be easily
accessed. DNA and protein sequences of billions of genes can be found and one can compare a sequence
to the many others within the databases. One very important entity responsible for this information as
well as being widely recognized globally is the National Center for Biotechnology Information (NCBI).
The website where one can access all their databases is: <http://www.ncbi.nlm.nih.gov/>. Please keep
note of this as it will be extremely useful throughout this coming term.
One of the NCBI’s goals is to use electronic databases and analytical tools to deal with the
overwhelming volume of data acquired in the process of understanding molecular biology and its role in
health and disease. As a great resource for molecular biology information, available with public access,
the NCBI creates different databases, conducts research in computational biology, develops software tools
for analyzing genomic data, and disseminates the biomedical information attained.
17
Within the NCBI genome database, you can search for species whose complete genome has been
sequenced and annotated. The research articles that have contributed to the data stored with the NCBI are
stored in PubMed. Annotation of genomes specifies gene sequences (Open Reading Frames, ORFs).
Additionally, millions of individual gene sequences (or partial sequences) of non-sequenced organisms
are also deposited. These constitute the Nucleotide Database. And from the translated version of these
genes, a Protein Database is created as well. Other databases available at NCBI include those from the
Human Genome Project, CancerChromosomes, Biosystems, SNP, and even Taxonomy.
Another very important function that the NCBI website can offer is a program that can analyze
and compare two or more sequences (nucleotide or protein). Called the Basic Local Alignment Search
Tool (BLAST), this program allows one to search a query sequence against another sequence, or even an
entire database of stored sequences to find the closest matches and view the similarities between them.
BLAST has been covered by Biol 571 in Fall 2009.
MATERIALS
Computers with internet connections
PROCEDURE
For this exercise, you will be directed to the following online databases and will get familiar with
the functionalities of them:
PEC database (http://www.shigen.nig.ac.jp/ecoli/pec/index.jsp )
1. Go to the PEC database website. You will see “Genes Statistical Table” separating E. coli genes
into essential, nonessential and unknown categories.
2. Click the number “302” and you will see the first 50 essential genes.
3. Click any gene name will lead you to the detailed information page for that gene. At the end of
the page, both amino acid and nucleotide sequences of the gene are provided, with FASTA
formats optional.
4. Copy Fasta format of either amino acid or nucleotide sequence and past into the BLAST input
boxes of either DEG database site or NCBI BLAST site for homology search analysis.
DEG database (http://tubic.tju.edu.cn/deg/)
BLAST analysis within DEG database only search for homologous sequences or regions stored in this
database.
SCORE (bits): The value S′ is derived from the raw alignment score S in which the statistical properties
of the scoring system used have been taken into account. By normalizing a raw score using the formula:
a “bit score” S′ is attained, which has a standard set of units, and where K and lambda are the statistical
parameters of the scoring system. Because bit scores have been normalized with respect to the scoring
system, they can be used to compare alignment scores from different searches (NCBI). Alignment of
similar sequences gives high scores.
18
Expect value: According to NCBI, the E-value is a parameter that describes the number of hits one can
“expect” to see by chance when searching a database of a particular size. It decreases exponentially with
the score (S) that is assigned to a match between two sequences. Essentially, the E-value describes the
random background noise that exists for matches between sequences. For example, an E-value of 1
assigned to a hit can be interpreted as meaning that in a database of the current size, one might expect to
see one match with a similar score simply by chance. This means that the lower the E-value, or the closer
it is to “0”, the higher is the “significance” of the match. However, it is important to note that searches
with short sequences can be virtually identical and have relatively high E-value. This is because the
calculation of the E-value also takes into account the length of the query sequence. This is because shorter
sequences have a high probability of occurring in the database purely by chance (NCBI).
Interpreting E-values:
E ≤ 0.02
sequences probably homologous
E between 0.02 and 1
homology unproven but cannot be ruled out
E>1
most likely not homologous
(the length of the nucleotides for the alignment is also very important)
To find out if an essential protein or gene from E. coli is well conserved among a number of key
pathogens, find out how many of the key pathogens have this protein or gene as essential in the DEG
database.
BLAST analysis (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi )
You should be very comfortable with this from Biol 571 at CSU Fullerton.
From result of sequence alignment using your query sequence, you should record the beginning (exact
nucleotide number on genome) and end (exact nucleotide number on genome) of a gene of interest
(subject) in order to download the sequence from NCBI.
REFERENCES
Lesk, A. M. Introduction to Bioinformatics, 2nd Ed, 2006 Oxford University Press, New York.
Gil et al., 2004 paper to read
19
REPORT AND QUESTIONS (Exercise 3)
Your Name: _____________________
A. Report
Go to the PEC database website, find essential gene list page by clicking the 302 essential gene number.
Click (gene) Symbol to sort the essential genes alphabetically. Only 50 genes are show per page. Locating
the gene whose symbol is below your last name but is closest to your last name’s first few characters. List
the gene name and its protein sequence below this paragraph. Your assignment is to use this gene’s amino
acid sequence to BLAST genomes of following seven key pathogens: Staphylococcus aureus,
Streptococcus pneumoniae, Enterococcus faecalis, Haemophilus influenzae, Pseudomonas aeruginosa,
Salmonella enterica and Acinetobacter baumannii. Print the tBLASTn report into PDF version and send
me the electronic version of the report (only the first 20 pages)
B. Questions:
1.
What is the definition of E-value?
2.
What E-value range is considered probably homologous? Which E-value will deem probably
not homologous?
3.
How many bacterial species are listed in DEG database whose essential gene lists are
published?
4.
Name two methods or approaches used to identify bacterial essential genes.
20
EXERCISE 4. Primer Design
OBJECTIVE


To understand the principles of primer design
To be familiar with primer design for various purposes
INTRODUCTION
An oligonucleotide is a short (vs long stretch strand of a duplex in a DNA fragment) stretch (around 2030) of typically deoxyribonucleotides. Oligonucleotides have many applications in molecular biology in
manipulation of genes and analysis of genes and their expression. Oligonucleotides serving to bind
specifically to a DNA fragment are called oligo probes (probes); these are useful in hybridization and
microarray experiments. More often, oligonucleotides of particular sequence (and orientation 5’ – 3’)
serve as “primers” for polymerase chain reactions (PCR) or primer extension reactions. These are called
primers. Designing primers is one of the most important basic tools of molecular biology for biomedical
research. Many criteria are to be met if specific and useful primers are to be designed. Optimal primer
sequences and appropriate primer concentrations are critical for efficient PCR or other reactions.
For PCR purposes, primers usually range between 15 and 30 bases in length and are designed to
flank the region to be amplified. Ideally, primers should possess G+C content of 40-60% and should not
contain sequences which could produce intra-molecular and inter-molecular base pairing of more than 3
bases. Ideally, the primer pair should have identical or similar melting temperature and this can be
achieved by adjusting the length of one of the primer.
The sequence of the primers could include non-base-paring sequences to facilitate cloning of the
PCR products into cloning vectors. These include designing recognition sites for restriction
endonucleases which are compatible to cloning vectors (and for directional cloning) and ligationindependent cloning (LIC) sequence for LIC strategy (see Exercise 5).
The following table provides detailed guidelines for design and use of primers:
Primer3 (http://frodo.wi.mit.edu/primer3/ ) is the most widely used primer design tool available
free of charge in the internet.
MATERIALS
Computer with access
to internet.
PROCEDURE
(PERIODS DEFINED)
Finding Gene
Sequences.
1. Go to the NCBI
website:
<http://www.nc
bi.nlm.nih.gov/
>
(from Qiagen PCR Brochure 08/2007)
21
2. Search for your assigned or desired gene or gene accession number under the “Nucleotide”
Database (using the pull-down button to select nucleotide).
3. Alternatively, you may search a gene from a genome page by typing in the name of the genome.
Try type in “ Acinetobacter baumannii ATCC 17978”. An Record No (NC_009085) appears.
Clicking the number, you will see the table below:
Genome Info:
Features:
BLAST
homologs:
Links:
Review Info:
Refseq:
NC_009085
Genes:
3453
COG
Genome Project
Publications: [1]
GenBank:
CP000521
Protein
coding:
3351
TaxMap
Refseq FTP
Refseq Status:
PROVISIONAL
Length:
3,976,747 nt
Structural
RNAs: 84
TaxPlot
GenBank FTP
Seq.Status:
Completed
GC Content:
38%
Pseudo
genes: 18
GenePlot
BLAST
Sequencing center:
Yale University
% Coding:
72%
Others: 1
gMap
TraceAssembly
Completed:
2007/03/07
Topology:
circular
Contigs:
None
CDD
Organism Group
Molecule:
DNA
Other genomes
for species: 23
4. If you click the GenBank Accession No. the GenBank annotated record (including the entire
sequence) will appear.
5. Within the record, you may search for a gene encoding a product by typing the enzyme name or a
gene name.
6. Click the CDS (CoDing Sequence) of the gene of your interest, which will generate only the
region of the genome specified by the CDS.
7. You may change the region shown by inserting the nucleotide positions in the window
located on the top-right corner (see example below): Extend the view by changing the
region shown (see right hand panel) by roughly 30 nucleotides on each end.
Helpful Tip:
Remember that the amino acid
methionine, which is encoded by
ATG, is always at the start of every
protein sequence and the stop codons
are TAA, TAG, or TGA.
8. You can go to FASTA format of the sequence by clicking “FASTA” button above the
record.
9. Print out or save the sequence in word document, and highlight the start and stop codons.
22
10. Sometimes, a gene’s coding sequence is located on the other strand relative to the
genome number system (meaning it starts at a higher number than its stop codon
number). Under such situation, a term “complement” should appear after CDS but before
the actual range of genome location numbers (in parentheses), such as the following:
gene
CDS
complement(7680..9611)
/locus_tag="A1S_0007"
complement(7680..9611)
/locus_tag="A1S_0007"
/codon_start=1
/transl_table=11
/product="putative transport protein"
/protein_id="ABO10504.2"
/db_xref="GI:193075927"
11. In such case, after you click the CDS hyperlink which will open the annotated gene
report, you can simply expand the “Customize View” window on the right side and check
the box “Show Reverse Complement” below Display Options and click Update View.
The gene ORF (open reading frame) sequence now starts with a start codon and ends
with a stop codon.
The primers that are desired for this term-project are to be used in conjunction with Novagen’s
pET30 Ek/LIC Cloning Kit. Please see Exercise 5 for an in-depth explanation of Ligation-Independent
Cloning.
Conventional primers are taken from a set of nucleotides within 100 bp outside the coding gene
sequence. Primer3 can help indicate which primers would be suitable based on GC nucleotide content and
melting temperature.
Designing the Primers for Ek/LIC Use.
12. Primers for your specified gene will include the following sequences (see Figure below):
Forward primer: 5’ - GAC GAC GAC AAG ATG … - 3’
Reverse primer: 5’ - GA GGA GAA GCC CGG TXX … - 3’
13. The ellipses will include at least 15
nucleotides (preferably 18-21) of the
gene:
Forward primer – first 18-21
nucleotides of the gene
Reverse primer – 15 nucleotides,
in reverse order, complementary to the
last 18-21 nucleotides of the gene
14. Print these primers out.
23
REPORT AND QUESTIONS (Exercise 4)
Your Name: _____________________
A. Report
Continue from last exercise’s report: you have obtained the protein blast (tBlastn) results of your assigned
essential gene product. Now for this exercise’s report, you are to design a pair of primers to amplify the
gene homolog from Acinetobacter baumannii ATCC 17978 strain. Specifically, you need to identify the
gene from this bacterium in the genbank database which is homologous to the PEC database essential
gene assigned to you (based on your last name). Find the open reading frame of the gene and design a pair
of PCR primers which will be used to amplify the gene and clone into a plasmid vector shown in the map
below. You have to design one or two restriction enzyme recognition sites into the primers to facilitate
cloning, assuming the vector can be digested at will based on the available unique restriction sites located
in the multiple cloning sites (MCS). To maximize restriction digest efficiency, you should also include 34 extra random bases beyond the restriction site sequences on the 5’ ends of both primers. In this report,
you are to paste in the gene sequence with start and stop codons highlighted and labeled plus the forward
and reverse primer sequences designed.
B. Questions.
1.
Define GC content. What is the proper range of GC content for PCR primers?
2.
What is optimal annealing temperature for a PCR primer pair with Tm of 54 °C and 55 °C?
3.
What to avoid in designing PCR primers for optimal PCR reactions (name four situations to
avoid)?
24
EXERCISE 5. Ligation-Independent Cloning
OBJECTIVE



Understand the strategy of LIC
Understand DNA transformation
Become familiar with LIC procedures
INTRODUCTION
Ligation-independent cloning (LIC) is a method used to create a recombinant plasmid
without the use of a ligase in vitro. Instead, we will use the power of hydrogen bonds and the
interal ligase of the competent cell to ensure that our recombinant plasmid is whole.
In LIC, recombinant enzymes are not even used to create the sticky ends either. The
primers that we designed in Exercise 4 contain the necessary LIC sequences for our purpose. The
PCR will be used to make multiple copies of our desired genes. The gene fragments will be
treated by T4 DNA polymerase to generate long “sticky ends” compatible to those of the linear
vector (prepared by the manufacturer) and inserted into a vector, pET30 ek/LIC (abbreviated as
pET30), via LIC and transformed into competent cells (called NovaBlue Gigasingles) by heat
shock. Once transferred into the cell, the gap between the DNA strands will be repaired (ligated)
by an internal ligase to form the recombinant plasmid.
Later on, we will transfer the plasmid (via heat shock or electroporation, depending on
the type of competent cells) to E. coli strain DL21BE3, an expressing host cell, in order to be
over-expressed by using an inducer isopropyl β-D-thiogalactopyranoside (IPTG). The fusion
proteins formed from the insertion of the gene into pET30 will contain an N-terminal histidine-6tag, which is exploited in the protein purification process. Protein purification is done by
immobilized divalent nickel adsorption chromatography. Once these proteins are purified, they
can then be concentrated and used for future assays.
Traditionally, inserting the PCR product into the plasmid requires the use of restriction
enzymes to generate sticky ends for the plasmid and gene to anneal with and ligases to ligate the
DNA molecules together. Instead, ligation-independent cloning (LIC) uses the 3’→ 5’
exonuclease activity of T4 DNA polymerase to generate specific sticky ends, which are
complementary to the overhangs present on the provided plasmid. The insert and the plasmid
will anneal together and the actual ligation occurs within the cell, after heat shock transformation
into competent E. coli, to provide the recombinant plasmid. The plasmid contains a kanamycin
resistant gene. Therefore, successfully transformed cells can be verified by plating on Difco
Luria-Broth and kanamycin (30 µg/ml) (LB+Kan) agar plates1.
The process of over-expression begins with cloning the desired gene into a plasmid that is
inducible by IPTG. The plasmid pET30 is such a plasmid and is used in this experiment. pET30
contains a lacI+ repressor gene, and there is a T7 promoter (recognized by the T7 DNA
polymerase) linked to a lac operator sequence which is where the lac repressor binds to in the
absence of lactose. The lac repressor prevents the RNA polymerase from transcribing the gene.
The standard cells (expression host), E. coli strain DL21BE3, contain a chromosomal copy of the
T7 RNA polymerase gene under the control of the lacUV5 promoter1. IPTG2 is a synthetic
analog of lactose and will inactivate the lac repressor to induce the expression of T7 DNA
polymerase and the LIC cloned gene located downstream of the T7 promoter on the pET30
plasmid.
25
Protein over-expression begins when the bacteria is induced with IPTG. The repressor is
inactivated, allowing more transcription to occur. The greater amount of mRNA produced will
result in more protein being translated. This experiment will also display whether the amount of
IPTG and the incubation period have a significant effect on the protein over-expression.
To obtain a protein extract, the cell must be lysed and all other macromolecules
destroyed. This calls for a lysozyme, DNase, and RNase to destroy the cell membrane, DNA, and
RNA, respectively. A phosphate-buffered saline solution is used as a buffer for all the reactions
to take place. Triton X-100 is nonionic detergent to remove the membrane proteins as well as to
neutralize the lysozyme reactions. Overexpression will be confirmed by polyacrylamide gel
electrophoresis (PAGE)3.
Initially, the recombinant plasmid pET30 generates a fusion protein with an N-terminal
histidine tag. This single His-6-tagged protein is in an exposed and relatively flexible site, which
allows the protein to be purified via metal chelate affinity chromatography using a charged
nickel resin. The negatively-charged histidines will bind to the positively-charged nickel on the
resin while other proteins will flow through. The target protein can then be eluted with a high
concentration of imidazole, which has a higher affinity for the divalent nickel ions than histidine.
EDTA is used in the strip buffer, because as a chelator itself, it would interfere with the metal
chelate chromatography. It also strips the Ni2+ from the resin4,5. Fusion protein over-expression
and purification will be performed in Exercise 13.
MATERIALS
pET30 ek/LIC cloning kit
Genomic DNA isolated and concentration determined
Waterbaths of 75 °C, 42 C
8 LB agar plates with kanamycin (30 g/ml)
polystyrene round-bottom tubes (Falcon® tubes) 1 tube per group (4 tubes for lab)
PROCEDURE (PERIODS DEFINED)
LIC (from Novagen’s Ek/LIC Cloning Kit with pET30)
T4 DNA Polymerase treatment of target insert (from PCR reaction in Exercise 7)
26
1. Assemble the following components in a sterile 1.5 ml microcentrifuge tube
kept on ice.
Table 2. Reaction mixture for T4 DNA polymerase treatment
Component
Purified PCR product (at least 0.2 pmol) + ddH2O (DNase/RNase
free)
10x T4 DNA Polymerase Buffer
dATP (25 mM)
DTT (100 mM)
T4 DNA Polymerase (2.5 U/µl, 0.5 unit per 0.1 pmol PCR product)
Total reaction volume
Volume (µl)
14.6
2
2
1
0.4
20
2. Start the reaction by adding the enzyme. Stir with the pipet tip to mix and
incubate at 22ºC for 30 minutes.
3. Inactivate the enzyme by incubating at 75ºC for 20 minutes.
4. Prepared Ek/LIC insert may be stored for months at -20ºC.
Annealing the Vector and Ek/LIC Insert
1. Assemble the following components in a sterile 1.5 ml microcentrifuge tube.
Component
pET30
T4 DNA Polymerase treated Ek/LIC insert (0.02 pmol)
Incubate at 22ºC for 30 minutes, then add
EDTA (25 mM)
Total volume
Volume (µl)
1
2
1
4
2. Mix by stirring with the pipet tip and incubate at 22ºC for 15-30 minutes.
Transformation into NovaBlue GigaSingles (Heat shock)
1. From the -80ºC freezer, remove 1 competent cell tube per gene, and place
them immediately on ice (leaving all but the cap immersed). Allow the cells to
thaw for 2-5 minutes. Each tube contains 50 l of competent cells.
2. Gently flick the tube 1-2 times to evenly resuspend the cells
3. Add 1 µl of the annealing reaction to the cells. Stir gently to mix.
4. Incubate on ice for 5 minutes.
5. Heat the tubes for exactly 30 seconds in a 42ºC water bath. Do not shake.
6. Place the tubes on ice for 2 minutes.
7. Add 250 µl of room temperature SOC medium.
8. Transfer to a 14 ml polystyrene round-bottom tube (Falcon® tube), which
allows for aeration to promote growth.
9. Incubate at 37ºC while shaking at 250 rpm for 60 minutes. Meanwhile, warm
your LB+Kanamycin (30 µg/ml) plates in the incubator.
10. On one plate, plate 10 µl of the cells; on a second plate, plate 100 µl of the
cell culture.
11. Set plates on the bench for several minutes to allow excess liquid to be
absorbed, and then incubate overnight at 37ºC.
Spread Plate Technique
1. Center the Petri dish on the turntable.
2. Pipet the sample onto the center of the agar
27
3. Use a sterile spreader to spread the sample over the entire surface of the agar
by placing the spreader flush along the agar and turning the Petri dish.
REFERENCES
1. Novagen. Ek/LIC Cloning Kits User Protocol TB163 Rev. G 1005. Darmstadt,
Germany: EMD Biosciences, Inc., 2004.
2. Fermentas Life Sciences. Catalog, Reagents: IPTG, dioxane free. Electronic document,
accessed Dec. 7, 2006. http://www.fermentas.com/catalog/reagents/iptg.htm.
3. Rybicki, E. & Purves, M. SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE).
Electronic document, accessed Dec. 6, 2006. http://www.mcb.uct.ac.za/sdspage.html.
4. Novagen. His•Bind® Kits User Protocol TB054 Rev. F 0106. Darmstadt, Germany:
EMD Biosciences, Inc., 2006.
5. Protocol 7: Purification of Histidine-tagged Proteins by Immobilized Ni2+ Adsorption
Chromatography. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor,
New York: Cold Spring Harbor Laboratory Press, 2001. p 15.44-15.48.
28
REPORT AND QUESTIONS (Exercise 5)
Your Name: _____________________
A. Report.
From the report of Exercise 4, you should have obtained the gene ORF sequence of A. baumannii ATCC
17978 derived from your assigned essential gene from PEC database. Please design a pair of primers to be
used to clone the gene using LIC strategy. In your report, you are to paste in the gene sequence with start
and stop codons highlighted and labeled and list the LIC primers (identify forward and reverse primers).
B. Questions.
1.
What is the role of T4 DNA polymerase in LIC procedures?
2.
We now know that isolated and purified pUC18 vector DNA molecule following double
restriction enzyme digestion by HindIII and EcoRI can form inter-molecular base-pairing
because every vector molecule has one unique HindIII sticky end and one EcoRI sticky end.
Can two DNA molecules of linearized (by the manufacturer) LIC vector form inter-molecular
base-pairing?
29
EXERCISE 6. Gram Negative Bacterial Genomic DNA Isolation
OBJECTIVE


Understand the concept of genomic DNA.
Understand the principle and practice of chromosomal DNA isolation from a bacterial cell.
INTRODUCTION
To isolate bacterial genomic DNA, one has to break the cell wall and cell membrane. Several
techniques for disrupting cell walls can be carried out at the bench either by EDTA-lysozyme lysis,
sonication, thermal incubation, glass-bead grinding or with a French pressure cell prior to isolation of
genomic DNA.
Both gram-positive and gram-negative cell walls contain a peptidoglycan layer, a covalent
multilayered structure of rigid glycan chains crosslinked by flexible peptide bridges. The glycan chains
are composed of two amino sugars N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc)
connected by β-1,4 glycosidic bonds with crosslinking peptides between the glycan layers. The quantity
and thickness, the length distribution, and the degree of crosslinking in gram-positive cells are more
extensive than in gram-negative cells. The lysis methods for gram-positive bacteria must overcome these
factors that result in a strong peptidoglycan barrier in order to cause disruption of the cell wall. A mixture
of lysozyme (an enzyme that hydrolyzes the β-1,4 glycoside linkages between GlcNAc and MurNAc of
the glycan backbone) and lysostaphin is used to efficiently lyse gram-positive cells. For this lab course,
we will need to isolate and purify genomic DNA from a gram negative bacterium: Acinetobacter
baummannii. Due to the limited quantities of genomic DNA required for amplification of target genes in
this course, we will employ a small scale (miniprep) chromosomal DNA isolation kit from Promega.
Specifically, genomic DNA is isolated in four steps: (1) Cell lysis. (2) RNase digestion. (3)
Cellular proteins removal by salt precipitation, leaving the high molecular weight genomic DNA in
solution. (4) Chromosomal DNA concentration and desalting by isopropanol precipitation.
MATERIALS
Per group of 5 students:
 Per group: one LB agar plate [4 LB agar plates for lab]
 1.5 ml microcentrifuge tubes (sterile)
 Microcentrifuge
 Acinetobacter baumannii ATCC17978 culture (4 tubes of overnight culture prepared by students)
 Water bath, 80°C
 Water bath, 37°C
 Water bath, 65°C
 Bench top vortex
 Isopropanol (Room temperature)
 70% ethanol (Room temperature)
 Pipets (P20, P200, P1000) and pipette tips (sterile)
 Ice
 Gloves
 Nuclei Lysis Solution (from Promega’s Wizard Genomic DNA Purification kit. Cat.# A1120)
 RNase solution (from Promega’s Wizard Genomic DNA Purification kit. Cat.# A1120)
 Protein Precipitation Solution (from Promega’s Wizard Genomic DNA Purification kit. Cat.#
A1120)
30
PROCEDURE (PERIODS DEFINED IF MORE THAN ONE DAY IS NEEDED)
(The following are performed based on groups, not individually)
Period 1 (Day1)
1. Streak the LB agar plate with a plate grown with A. baumannii ATCC 17978
Period 2 (Day2)
1. Transfer 3 ml of sterile LB broth medium into a Falcon tube aseptically.
2. Inoculate a single colony from a streaked A. baumannii plate into a Falcon plastic tube containing 3
ml of LB broth
3. Incubate overnight at 37C.
Period 3 (Day 3)
1. Every group of 3 students will transfer 1 ml of an A. baummannii ATCC17978 broth overnight
culture into a 1.5 ml microcentrifuge tube using P-1000 pipettes.
2. Centrifuge the cells in a microcentrifuge for 2 min at 10,000 rpm to pellet the cells. Remove
supernatant.
3. After removal of supernatant, gently resuspend cell pellet in 600 l Nuclei Lysis Solution using the
pipet and a tip.
4. Incubate at 80°C for 5 minutes to lyse the cells. Cool to room temperature on bench.
5. Add 3 l RNase and mix by inverting 2-5 times.
6. Incubate at 37°C for 30 minutes. Cool the sample to room temperature.
7. Add 200 l of Protein Precipitation Solution to the RNase-treated cell lysate. Vortex vigorously at
high speed for 20 seconds.
8. Incubate the sample on ice for 5 minutes.
9. Centrifuge lysate in a microfuge for 3 min at 10,000 rpm. While waiting add 600 l room
temperature isopropanol to a 1.5 ml microcentrifuge tube.
10. Transfer the supernatant containing the DNA to the 1.5 ml microcentrifuge tube containing 600l
isopropanol. (When transferring the supernatant some liquid may remain in the original tube
containing the protein pellet. Leave this residual liquid as to avoid contaminating the DNA solution
with precipitated protein.)
11. Gently mix by inversion until you see the precipitated thread-like strands of DNA forming a mass.
12. Centrifuge at 10,000rpm for 2 minutes.
13. Carefully pour off the supernatant and drain the tube on clean absorbent paper. Add 600 l of room
temperature 70% ethanol and gently wash the DNA pellet by inverting the tube several times.
14. Centrifuge at 10,000 rpm for 2 minutes. Carefully aspirate the ethanol with a pipette.
15. Drain the supernatant on clean absorbent paper and let the pellet air-dry for 15 minutes.
16. Add 100 l of DNA Rehydration Solution to the tube and rehydrate the DNA by incubating at 65°C
for 1 hour. Mix the solution periodically by gently tapping the tube.
17. Store the DNA at 4°C.
18. Concentration determination by Nanodrop spectrometer.
REFERENCES
Wizard Genomic DNA Purification kit manual (Promega)
Mahalanabis M., et al. (2009). Cell lysis and DNA extraction of gram-positive and gram-negative bacteria
from whole blood in a disposable microfluidic chip. Lab Chip. Vol. 9, 2811-2817.
31
REPORT AND QUESTIONS (Exercise 6)
Your Name: _____________________
A. Report.
Below paste in your data/result of genomic DNA concentration determination. If your DNA concentration
is unusually low or you did not see DNA precipitation occurring during the isolation/purification
procedure, explain the possible cause of such results and observation.
B. Questions.
1.
What is the role of RNase during the procedure of DNA isolation?
2.
Why did we use a step of DNA precipitate wash using 70% ethanol but not 100% ethanol?
3.
Why do we not vortex the purified DNA in the DNA rehydration solution to dissolve it
quickly?
32
EXERCISE 7. Polymerase Chain Reaction
OBJECTIVE


To understand the history of PCR and its contribution to the molecular biology, biomedicine and
biotechnology
To be familiar with procedures and critical factors of PCR
INTRODUCTION
The invention of the polymerase chain reaction (PCR) by Kary Mullis and colleagues in mid 1980s
revolutionized molecular biology and biomedical research. They described in vitro amplification of
single-copy mammalian genes using E. coli’s Klenow fragment (part of DNA polymerase I). The
subsequent use of a thermostable polymerase from Thermus aquaticus greatly improved the efficiency of
the PCR reactions and paved the way for the automation of the method now known as thermocyclers.
By the end of the 1980s, molecular cloning was no longer the only way of isolating genes. PCR
had become an indispensable method in molecular biology and biomedical research. The basic principle
of PCR is to generate identical copies of a duplex DNA molecular via repeated (template DNA)
denaturing, (specific primer) annealing (to strands of DNA templates) and extension, exponentially.
Due to required denaturing of template DNA, thermostable DNA polymerases are essential to PCR.
Additional improvements and variation of PCR methods have also been developed for various purposes:
Long PCR, Overlap PCR, Inverse PCR and quantitative PCR.
The basic PCR reactions contain seven essential components: a thermostable DNA polymerase, a
pair of synthetic oligonucleotides (primers), deoxynucleoside triphosphates (dNTPs), divalent cations
(required by all thermostable DNA polymerases), buffer to maintain pH, monovalent cations and template
DNA. To simplify routine PCR experiments, vendors market various PCR Master Mix kits in which all
components of the reaction, except template and primers, are included in defined higher concentrations
(usually 2X or 2.5 X of the final required concentrations).
In this course, we will use a PCR Master Mix kit from 5 Prime using Taq polymerase. 5 PRIME
MasterMix is a 2.5x concentrated, ready-to-use reagent mix for the amplification of nucleic acid
templates using PCR and is supplied with an additional magnesium chloride solution. Due to the 2.5x
concentration of the 5 PRIME MasterMix, most (60 %) of the final reaction volume can be used for the
addition of primer, template and other additives. One ml of the 5 PRIME MasterMix is sufficient for 100
amplification reactions of 50 l. 5 PRIME MasterMix contains TaqMaster PCR enhancer which increases
yield and robustness against PCR inhibitors such as Humic acid and blood compounds. The 2.5x 5
PRIME MasterMix contains Taq DNA Polymerase (62.5 U/ml), 125 mM KCl, ®-CA360 in 0,5%, 500
M of each dNTP, 75 mM Tris-HCl pH 8.3, 3.75 mM Mg(OAc)2 , 0.25 % Igepal and stabilizers. This
corresponds to final concentrations in the PCR reaction of 1.25 U Taq DNA Polymerase, 50 mM KCl, 30
mM Tris-HCl, 1.5 mM Mg 2+, 0.1% Igepal®-CA360 and 200 M of each dNTP. PCR reactions that
require higher than 1.5 mM Mg2+ concentrations can be optimized with the separate 25 mM Mg2+ solution
provided.
MATERIALS
Template DNA (isolated in Ex. 6)
Primers (LIC specific)
DNase-free/RNase-free dI water (Invitrogen)
Thermocycler (Eppendorf)
0.2 ml PCR tubes (Fisher ?)
5 PRIME Master Mix (Fisher Scientific)
33
PROCEDURE (PERIODS DEFINED)
Before starting, mix the 5 Prime MasterMix thoroughly to avoid localized differences in salt
concentrations.
1. Prepare the template/primer mix in a 0.2 ml PCR tube by adjusting the total volume to the values
given in Table 1 below with DNase-free/RNase-free dI water.
2. Dispense the appropriate volume of the 5 PRIME MasterMix into a PCR tube (e.g. 20 l for a 50
l PCR reaction).
3. Add the template/primer mix to the PCR tube containing the 5 PRIME MasterMix. Close the tube
and mix well, avoiding formation of foam. If necessary, centrifuge briefly and place tube on ice.
4. Start the PCR program on a thermal cycler. To suppress amplification of nonspecific PCR
products, the thermal cycler should be preheated (>90°C) before placing the PCR tube(s) on the
cycler block.
5. If necessary, PCR reactions can be optimized simply by increasing the volume of 5 PRIME
MasterMix added per reaction. Different values and corresponding final concentrations of the
major reaction components for 50 l reactions are shown in table 2 as a guideline.
34
6. For initial PCR experiments before groups design their own primers for selected gene targets, a
pair of primers used in Dr. Xu’s lab in the past will be used:
AB fabI FAFABITAG1F (42-mer); Tm= 68.6 °C; MW=12987.6
5’ GAC GAC GAC AAG ATG CAT TAT GCA ATTT…3’
stock: 3333 ng/uL
AB fabI R –
AFABITAG1R (45-mer); Tm=72°C; MW=13835.2
5’ GAG GAG AAG CCC GGT TT ATT CATCATCC…3’
stock: 3322 ng/uL
7. These primers were designed for LIC strategy, thus they contain LIC specific sequences (see
underlined sequences above).
8. The fabI gene’s length is 874 bp. Adding the extra bases on the PCR product from the LIC
sequences will produce a product with an estimated size of 901 bp.
9. Routinely, the annealing temperature should be 5 °C lower than the Tm’s of the primers and the
primers’s Tm values should be as close as possible.
10. PCR will be run with the following cycling parameters:
Segment
Number of
cycles
Temperature
Duration
1
2
1
30
95°C
95°C
2 minutes
30 seconds
54 ºC (5 ºC below Tm)
30 seconds
72°C
72°C
4°C
1 minute
10 minutes
∞
3
4
1
1
REFERENCES
5 PRIME MasterMix Manual.
Sambrook and Russel. 2001. Molecular Cloning. 3rd ed. Cold Spring Harbor Laboratory Press. Cold
Spring Harbor, NY.
35
REPORT AND QUESTIONS (Exercise 7)
Your Name: _____________________
A. Report.
Below paste in your data/result of PCR experiments. Include agarose gel photos and labels of various
lanes. Additionally, specify the template DNA and the PCR primers used. If one or more PCR
experiments did not work properly, please discuss the possible reasons.
B. Questions.
1.
Describe PCR reaction conditions/parameters in layman’s terms.
2.
What is the range of template DNA (in quantity) to be used if the gene to be amplified is
located on a chromosome?
3.
Why there is a step of 10 min incubation at 72 °C after the 30 cycles during PCR?
36
EXERCISE 8. Agarose Gel Electrophoresis
OBJECTIVE


Understand the concept of nucleic acid gel electrophoresis
Become familiar with agarose gel electrophoresis
INTRODUCTION
Nucleic acids of different lengths migrate at distinct rate on agarose or polyacrylamide gels. This forms
the basis for separation of nucleic acids (both DNA and RNA). While polyacrylamide gels are used for
the separation and resolution of very small DNA fragments (10 - 500 nucleotides), agarose gels are the
most routine matrix to separate larger DNA fragments such as plasmids or genomic DNA fragments (in
Pulsed Field Gel Electrophoresis). The concentration of the gels also determines the rate of migration for
nucleic acids. Higher density gels present more obstacles for larger fragments. Thus, for separation of
large fragments, a gel with a low concentration of the matrix is desirable; and the reverse is also true.
Separated nucleic acids can be visualized on a gel by staining them with intercalating fluorescent dyes.
Ethidium bromide is the most commonly used dye for visualization of DNA under UV (ultra-violet) light.
Separation and analysis of DNA fragments by agarose gel electrophoresis is one of the most common
techniques that is used daily in molecular biology laboratories. In this exercise, students will run a miniagarose gel to visualize the DNA molecules from their plasmid isolation and restriction digest. This
method will also be used for confirm the presence and yield of amplified DNA fragments from PCR
experiments (Ex. 6)
MATERIALS
Per table:
 1 agarose gel tray
 Mini-gel apparatus
 1x TAE running buffer
 Power supply
 1.7 ml microcentrifuge tubes
 10 x DNA sample loading dye
 Pipets (P10 or P20) and pipette tips
 DNA ladder (from Fermentas or another vendor)
 UV safety glasses
 UV transilluminator
 Polaroid gel camera and films or a digital camera
 Gloves
 Ethidium bromide solution for gel staining (containing ethidium bromide; carcinogenic, BE
CAREFUL, must use gloves)
PROCEDURE (PERIODS DEFINED)
1. Each group of students will prepare an agarose gel using 1X TAE running buffer. Place the gel into
the mini-gel apparatus submerged in 1X TAE running buffer.
2. Add 2 l of the 10 x DNA sample loading dye to each of the tubes containing 5 l of PCR product
and 13 l ddH2O.
37
3. Using a pipet (P20), carefully load 10 l of 1 kb DNA ladder (provided) to the first well for each
group.
4. Using a pipet, mix the contents of each tube and carefully load 20 l of the tube contents into the
other wells. Change tips between samples. Make sure to write down the well #s and the
corresponding samples (e.g., well#2 contains plasmid control, no enzyme) for each well in your
notebook. During the process of loading DNA, carefully lower the pipette tip below the surface of
the buffer and directly over the well, and slowly dispense the sample. Do not penetrate the bottom
of the well with the pipette tip. The loading dye contains glycerol which facilitates sinking of the
samples into the bottom of the wells.
5. Secure the gel apparatus with its cover, making sure DNA samples are running toward the electrode
connected to the red wire. Connect the electrodes to a power supply (black to black and red to red),
turn on the power supply, and run the gel at 110 V until bromophenol blue dye is approximately
near the bottom of the gel. This will take about 1 hr.
6. After electrophoresis is completed, turn off the power supply. Wearing gloves, place the gels in the
ethidium bromide solution in a plastic box with slow shaking for 20 min. Place the gel on the UV
transluminator. DNA fragments and their relative migrated location can be visualized by turning on
the UV light; make sure the transparent cover of the UV transilluminator is down, protecting
students from UV exposure. When taking a picture of the gels for analysis, it is necessary to lift the
UV transilluminator cover and wear a full face protector during the course of brief UV exposure.
[Dispose of the gel containing ethidium bromide only in a special designated container. Do not
dispose of the gel in the regular trash.]
REFERENCES
38
REPORT AND QUESTIONS (Exercise 8)
Your name:______________________
A. Report
Affix the picture of the agarose gel showing your PCR fragment size and purity along with DNA ladder.
B. Answer the following questions:
1. Which electrode (cathode or anode) does DNA migrate toward?
2. Why does one have to add loading dye for agarose gel electrophoresis?
3. What is the role of ethidium bromide in agarose gel electrophoresis and what is the mechanism?
4. Why is ethidium bromide dangerous to human?
39
EXERCISE 9. PCR Product Purification
(based on Qiagen’s QIAquick spin handbook)
OBJECTIVE


To understand the principles of DNA absorption to silica membrane and the subsequent
separation and removal of unwanted components (primers, enzymes and salts, which could
hinder the efficacy of the Ligation-Independent Cloning kit) from PCR reactions
To become familiar with the procedures of PCR product purification
INTRODUCTION
The QIAquick system combines the convenience of spin-column technology with the selective binding
properties of a uniquely designed silica membrane. Special buffers provided with each kit are optimized
for efficient recovery of DNA and removal of contaminants in each specific application. DNA adsorbs to
the silica membrane in the presence of high concentrations of salt while contaminants pass through the
column. Impurities are efficiently washed away, and the pure DNA is eluted with Tris buffer or water (see
Figure 1). QIAquick spin columns offer 3 handling options — as an alternative to processing the spin
columns in a microcentrifuge, they can now also be used on
any commercial vacuum manifold
with luer connectors.
The QIAquick silica membrane is uniquely adapted to
purify DNA from both aqueous solutions and agarose gels, and
up to 10 µg DNA can bind to each QIAquick column. The
binding buffers in QIAquick Spin Kits provide the correct salt
concentration and pH for adsorption of DNA to the QIAquick
membrane. The adsorption of nucleic acids to silica surfaces
occurs only in the presence of a high concentration of
chaotropic salts, which modify the structure of water.
Adsorption of DNA to silica also depends on pH. Adsorption is
typically 95% if the pH is ≤7.5, and is reduced drastically at
higher pH. If the loading mixture pH is >7.5, the optimal pH
for DNA binding can be obtained by adding a small volume of
3 M sodium acetate, pH 5.0.
Buffer PB in the QIAquick PCR Purification Kit
allows the efficient binding of single or double-stranded PCR
products as small as 100 bp and the quantitative (99.5%)
removal of primers up to 40 nucleotides. This kit can therefore
be used to remove oligo-dT primers after cDNA synthesis or to
remove unwanted linkers in cloning experiments.
During the DNA adsorption step, unwanted primers
and impurities, such as salts, enzymes, unincorporated
nucleotides, agarose, dyes, ethidium bromide, oils, and
detergents (e.g., DMSO, Tween® 20) do not bind to the silica
membrane but flow through the column. Salts are
quantitatively washed away by the ethanol-containing Buffer
PE. Any residual Buffer PE, which may interfere with
subsequent enzymatic reactions, is removed by an additional
centrifugation step.
Elution efficiency is strongly dependent on the salt
40
concentration and pH of the elution buffer. Contrary to adsorption, elution is most efficient under basic
conditions and low salt concentrations. DNA is eluted with 50 or 30 µl of the provided Buffer EB (10
mM Tris·Cl, pH 8.5), or water. The maximum elution efficiency is achieved between pH 7.0 and 8.5.
When using water to elute, make sure that the pH is within this range. In addition, DNA must be stored at
–20°C when eluted with water since DNA may degrade in the absence of a buffering agent. Elution with
TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0) is possible, but not recommended because EDTA may
inhibit subsequent enzymatic reactions.
MATERIALS
Ethanol (96-100%)
Microcentrifuges
1.5 ml microcentrifuge tubes
3 M sodium acetate, pH 5.0
DNase-free/RNase-free dI water (Invitrogen)
QIAquick PCR purification kit (Qiagen Cat No. 28104)
PROCEDURE
*Before beginning, make sure that ethanol (96-100%) has been added to Buffer PE.
Procedures:
1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
2. Place a QIAquick spin column in a provided 2 ml collection tube.
3. To bind DNA, apply the sample to the QIAquick column and centrifuge at 13,000 rpm
(17,900 x g) for 60 seconds.
4. Discard the flow-through.
5. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge at 13,000 rpm
(17,900 x g) for 60 seconds.
6. Discard the flow-through and centrifuge the column for 1 minute to remove any residual
ethanol.
7. Place the QIAquick column in a clean 1.5 ml microcentrifuge tube.
8. To elute DNA, add 50 µl Buffer EB to the center of the QIAquick membrane and
centrifuge the column for 1 minute.
9. Purified PCR DNA can be stored at -20 C for future use (such as LIC cloning procedures)
REFERENCES
Qiaquick Spin Handbook. Qiagen.
41
REPORT AND QUESTIONS (Exercise 9)
Your name:______________________
Answer the following questions:
1. Why do we need to purify PCR products?
2. In QIAquick PCR product purification kit, what type of material does the PCR DNA product bind
to?
3. What is the role of microcentrifuge in PCR product purification?
4. What is the optimal pH at which PCR products are eluted?
42
EXERCISE 10. Bacterial Transformation
OBJECTIVE



Understand the phenomenon of natural transformation
Understand the concept of transformation using artificially made competent bacterial cells
Become familiar with heat-shock transformation
INTRODUCTION
Transformation is the uptake by a recipient bacterium of foreign naked DNA molecules from an
environment (including culture medium) and incorporation of the foreign DNA into its own chromosome
in a heritable form. Certain bacterial species (such as Bacillus subtilis, Streptococcus pneumoniae and
Haemophilus influenzae) are naturally competent in adsorbing and integrating foreign genetic materials
into their own chromosomes. This phenomenon is called natural transformation or natural competence.
Many bacterial species which are not naturally competent can be made into competent for the
purposes of introducing heterogeneous genetic materials into the cells. For example, Escherichia coli, a
model bacterium, can be easily made competent chemically by treating with salt such as calcium chloride
or rubidium chloride. Competent cells are stored at -80 °C in aliquots and one or more tubes can be
thawed for transformation experiments using heat-shock at 42 °C. Alternatively, E. coli cells grown to
late log phase and washed in cold glycerol solution multiple times can become competent during an
electric shock when placed in a high voltage differential environment. These cells are called
electrocompetent cells and the process of introducing foreign DNA molecules into bacterial cells is called
transformation by electroporation.
In this class, heat-shock transformation procedures will be used. NovaBlue GigaSingles™ Competent
Cells (Cat. No. 71127) are provided in Ek/LIC Vector Kits and should be used for initial cloning with
pET30 ek/LIC vector. NovaBlue is a convenient cloning host because the recA endA mutations facilitate
high transformation efficiency and high yields of excellent plasmid DNA. NovaBlue GigaSingles
Competent Cells are provided in 50-μl single-use aliquots. The pET30 ek/LIC kit also contain competent
cells of expression host strains [E. coli DL21 (DE3)] in 0.2 ml aliquots (10 transformations).
MATERIALS
pET30 ek/LIC cloning kit
Annealed insert plus LIC vector or other recombinant plasmids
Waterbaths of 75 °C, 42 °C
LB agar plates with kanamycin (30 g/ml)
polystyrene round-bottom tubes (Falcon® tubes)
PROCEDURE
Transformation into NovaBlue GigaSingles (Heat shock) (already described in Ex. 5)
1.
From the -80ºC freezer, remove 1 competent cell tube per gene, and place them
immediately on ice (leaving all but the cap immersed). Allow the cells to thaw for
2-5 minutes. Each tube contains 50 l of competent cells.
2.
Gently flick the tube 1-2 times to evenly resuspend the cells
3.
Add 1 µl of the annealing reaction to the cells. Stir gently to mix.
4.
Incubate on ice for 5 minutes.
5.
Heat the tubes for exactly 30 seconds in a 42ºC water bath. Do not shake.
43
6.
7.
8.
9.
10.
11.
Place the tubes on ice for 2 minutes.
Add 250 µl of room temperature SOC medium.
Transfer to a 14 ml polystyrene round-bottom tube (Falcon® tube), which allows
for aeration to promote growth.
Incubate at 37ºC while shaking at 250 rpm for 60 minutes. Meanwhile, warm your
LB+Kanamycin (30 µg/ml) plates in the incubator.
On one plate, plate 10 µl of the cells; on a second plate, plate 100 µl of the cell
culture.
Set plates on the bench for several minutes to allow excess liquid to be absorbed,
and then incubate overnight at 37ºC.
Transformation into expression host E. coli DL21 (DE3)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
From the -80ºC freezer, remove 1 competent cell tube [DL21 (DE3)] (1 tube
containing 200 l of competent cells, which needs to be divided into 10 aliquots
for 10 transformation), and place them immediately on ice (leaving all but the cap
immersed). Allow the cells to thaw for 2-5 minutes.
Visually examine cells for thawing and gently flick the tube 1-2 times to
resuspend cells evenly. Never vortex competent cells.
Pre-chill required number of 1.5 ml micrcentrifuge tubes on ice. Pipet 20 l cell
aliquots into each pre-chilled tube.
(Optional) To determine transformation efficiecy, add 1 l (0.2 ng) test plasmid to
a tube containing cells. Gently flick tube to mix, Return to ice.
Add 1 µl of the recombinant plasmid prepared previously to the cells. Stir gently
to mix. Return to ice.
Incubate on ice for 5 min.
Heat tubes for exactly 30 s in a 42 C water bath. Do not shake.
Place tubes on ice for 2 min.
Add 80 ml room temperature SOC medium to each tube. Keep tubes on ice while
handling.
Transfer to a 14 ml polystyrene round-bottom tube (Falcon® tube), which allows
for aeration to promote growth.
Incubate at 37 C with shaking (250 rpm) for 60 min prior to plating on selective
medium.
On one plate, plate 10 µl of the cells; on a second plate, plate 100 µl of the cell
culture.
Set plates on the bench for several minutes to allow excess liquid to be absorbed,
and then incubate overnight at 37ºC.
REFERENCES
Novagen Technical Bulletin No. 163.
44
REPORT AND QUESTIONS (Exercise 10)
Your name:______________________
A. Report
Draw diagrams of transformant colonies on your transformation plates. Explain the difference in number
of colonies obtained.
B. Answer the following questions:
1.
Define transformation. What distinguish it from transduction?
2.
What would you expect if you forget to heat shock the cells after adding plasmid DNA or
annealed vector + insert?
3.
Search the web about “frederick griffith and transformation” and briefly describe what you
found in less than five sentences.
45
EXERCISE 11. Plasmid Miniprep
OBJECTIVE


Understand the concept of plasmid and plasmid vectors.
Understand the principle and practice of plasmid isolation from a bacterial cell.
INTRODUCTION
Plasmids are routinely used as cloning vectors in molecular biology laboratories. One of the most
important features of plasmid vectors is their ability to replicate independently of bacterial chromosome
(extra-chromosomal replication), permitting in vivo amplification of the cloned DNA for the purpose of
genetic manipulation. Another feature is that many plasmids carry antibiotic resistant genes whose
products confer characteristic resistant phenotype for the cells that have acquired the plasmid(s). In order
to use plasmid vectors for gene manipulation, it is necessary to isolate and characterize a plasmid from a
bacterial clone. In this exercise, students will isolate a plasmid from a bacterial culture, perform
restriction endonuclease digestion and visualize the plasmids and their digested products on an agarose
gel.
Over the years, a number of methods have been developed for isolating plasmids from
bacteria. A common requirement in all of these methods is the breaking of the cell wall and
denaturation of cellular proteins and the chromosomal DNA. Subsequently, plasmid DNA and
RNA are separated from cell debris and denatured chromosomal DNA by centrifugation.
Plasmid DNA can be further purified from the contaminating RNA molecules by RNAse
treatment. In this exercise, students will isolate plasmid via a miniprep procedure based on
alkaline lysis using a kit from QIAGEN. The procedure consists of three basic steps: (1)
preparation and clearing of a bacterial lysate; (2) adsorption of DNA onto the QIAprep
membrane; (3) washing and elution of plasmid DNA. All steps are performed without the use of
phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation.
MATERIALS
Per group of 3 students:
 1.7 ml microcentrifuge tubes (sterile)
 Microcentrifuge
 E. coli culture containing a plasmid with insert
 Pipets (P20, P200, P1000) and pipet tips (sterile)
 Ice
 Nuclease-free sterile deionized water
 Gloves
 Buffers P1, P2, N3, PE and EB (from QIAGEN)
 Spin column (from QIAGEN)
PROCEDURE
1. Every student will transfer 1.5 ml of an E. coli broth culture (harboring a plasmid) into a 1.7 ml
microcentrifuge tube using P-1000 pipets.
2. Centrifuge the cells in a microfuge for 2 min at 10,000 rpm.
3. After removal of supernatant, resuspend cell pellet in 250 l Buffer P1 using the pipet and a tip
(note, RNase and LyseBlue reagent have been added to Buffer P1 by instructional staff).
46
Add 250 l Buffer P2 and mix thoroughly by inverting 4-6 times.
Add 350 l Buffer N3 and mix by inverting 4-6 times.
Centrifuge the lysate at 13,000 rpm for 10 min.
Apply the supernatants from step 6 to the QIAprep spin column by decanting or pipetting.
Centrifuge for 30 – 60 seconds. Discard the flow-through.
Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuge for 30-60 s (note, the
instructional staff adds ethanol to Buffer PE prior to first use).
10. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
11. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 l
Buffer EB or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge
for 1 min.
12. Determine plasmid concentration using Nanodrop.
4.
5.
6.
7.
8.
9.
REFERENCES
Qiagen QIAQuick miniprep manual
47
REPORT AND QUESTIONS (Exercise 11)
Your name:______________________
A. Report
Describe your result of plasmid preparation including its concentration and restriction digest profile.
B. Answer the following questions:
1.
What do you expect in your plasmid miniprep experiment if the bacterial clone is grown
overnight without proper antibiotic selection (ie, in LB broth medium but with any
antibiotic)?
2.
If the plasmid you isolated is a pET30 ek/LIC vector (5.4 kb by itself) with a 1.5 kb insert
DNA, what restriction enzymes would you like to use to digest the plasmid in order to
confirm the size of the insert (a hint, check out the plasmid map on a Powerpoint slide)?
What approximate sizes in kilobases (kb) do you expect to see on an agarose gel if everything
works perfectly?
48
EXERCISE 12. Restriction Digest Analysis
OBJECTIVE



Understand the function of restriction endoncleases in bacterial cells
Understand the utility of these enzymes in genetic manipulation
Master single or double restriction enzyme digest of DNA molecules
INTRODUCTION
Many bacterial species produce restriction endonucleases (restriction enzymes). These enzymes are used
by the host cells as a defensive mechanism against invading foreign DNA. They recognize very specific
DNA sequences and make double stranded breaks on DNA at or near the recognized sites. These enzymes
normally do not cut the host DNAs due to either absence of the specific sequences or modification of
certain nucleotides on DNA that mask these sequences.
Restriction enzymes and their specific recognition DNA sequences are integral to the molecular
biology and manipulation of genetic materials. The natural presence or intentional insertion of specific
restriction recognition sites enable scientists to clone genes or gene fragments and to transfer genes and
their encoded traits into desired host cells or organisms. In this exercise, students will perform a
restriction enzyme digest on the plasmid isolated using two restriction enzymes, each of which recognizes
and cuts within a different DNA sequence. Your group should have successfully cloned an essential gene
into the LIC site (see figure below). To confirm the presence of an insert DNA of appropriate size,
restriction digest of the isolated plasmid (named pET30-YourGene-1) using appropriate restriction
enzymes (care to select two?) and subsequent agarose gel electrophoresis should confirm whether or not
your group has cloned a DNA and the size of the insert DNA.
MATERIALS
Each student:
 1.7 ml microcentrifuge tubes (sterile)
 Microcentrifuge
 Pipets (P20) and pipet tips (sterile)
 Ice
 BglII, BamHI, and their 10X buffers from New England Biolabs
 Sterile Dnase/Rnase free water
 Gloves
 Water bath (37ºC)
49
PROCEDURE
1. Each student would have isolated a miniprep plasmid DNA as described previously in Exercise 11.
2. Set up the following reactions after label your microtube with your initials, name of plasmid:
Component
Vol (l)
10X Reaction Buffer #3
2
Your plasmid DNA
7
Dnase/Rnase free water
10
BglII
0.5
BamHI
0.5
------------------------------------------------------------Total rection vol
20
3. Incubate the microtubes in 37ºC water bath for 45 min.
4. After the incubation, proceed to gel electrophoresis as in Exercise 8.
REFERENCES
50
REPORT AND QUESTIONS (Exercise 12)
Your name:______________________
A. Report
Describe your result of plasmid restriction digest and agarose gel electrophoresis.
B. Answer the following questions:
1.
What other restriction enzyme may be used along with BglII to digest your recombinant
plasmid to determine the size of the insert without worrying about the incompatibility of the
reaction buffer (assuming you have to use enzymes from New England Biolabs)?
2.
If KpnI has to be used, name one restriction enzyme which can share the same reaction buffer
with KpnI (assuming you have to use enzymes from New England Biolabs).
3.
If after your double enzyme digest, you observe three or more distinct DNA bands on the
agarose gel and the sum of the bands’ size equals the expected size of the recombinant
plasmid. What is your explanation and how would you find out whether your explanation is
correct?
51
EXERCISE 13. His-tagged Protein Over-expression and Purification
OBJECTIVE



Understand the principle of protein over-expression and his-tag aided purification
Understand the principle of protein separation
Become familiar with procedures of protein over-expression, purification and SDS-PAGE
INTRODUCTION
After target genes are successfully cloned into pET30 ek/LIC vector and the recombinant plasmids
transferred to expression host E. coli DL21(DE3), we will proceed with induction of gene expression for
the purpose of over-producing the encoded protein products (our selected essential drug targets). Once the
proteins are confirmed to be over-expressed, they have to be purified to near homogeneity for developing
enzymatic assays for screening compound libraries. As the vector map in the last exercise (Exercise 12)
indicates, the cloned gene is under the control of a T7lac promoter, located upstream of the fusion gene
which encodes an N-terminal His-tag (and an S-tag) followed by your selected protein sequence.
Eventually, if so desired, the purified his-tagged target protein can be further cleaved using a sequencespecific protease enterokinase (also known as enteropeptidase) at a site immediately upstream of the
target protein sequence. This may not be necessary if the his-tag does not interfere with the activity of the
enzyme.
Specifically, bacterial cells of certain growth stage (eg, following induction) are centrifuged and
the cell pellet is subject to harsh treatment to lyse the cells and release cellular contents including
proteins. Nucleases are added to digest DNA and RNA molecules, leaving proteins as the major
component of the cell-free extract. In this exercise, Novogen’s BugBuster Protein Extraction Reagent will
be used to obtain protein extracts from cell pellets. To determine if the protein of your interest is overexpressed among hundreds (if not thousands) of cellular proteins, Sodium dodecyl sulfate/polyacrylamide
gel electrophoresis (SDS-PAGE) will be employed. To perform SDS-PAGE, the protein sample is
combined with 2X SDS-PAGE loading dye and denature by heating at 95 C for 5 min before loading into
the wells of the precast protein gel.
After protein is confirmed to be over-expressed, the cell growth and induction will be repeated at
a larger scale to obtain sufficient quantities of proteins for assay development and HTS. Over-expressed
proteins will be purified using Novogen’s His-Bind Kit.
MATERIALS
Phosphate-buffered saline (PBS)
His-Bind purification kit (Novagen#70793)
Bug-Buster 10X Protein Extraction Reagent (Novagen #70921-3) to be diluted to 1X with
Phosphate-buffered saline.
Perfect Protein Markers 10-225 kDa (Novagen #69079-3
Isopropyl-beta-D-thiogalactopyranoside (Fisher#BP1755)
Phenylmethylsulfonyl fluoride (PMSF) (Pierce#36978)
Difco Luria-Bertani (LB) broth (BD#8120771)
Benzonase (Novagen)
250 ml Erlenmeyer Flask
Spectrophotometer
Fisher Pierce Precast SDS-PAGE gel (Fisher PI25201)
52
PROCEDURE
Cell growth and induction.
1. The frozen cell stocks [such as E. coli BL21(DE3) host] with proper recombinant plasmid
(eg., pET30 ek/LIC containing insert DNA) over-expressing a given protein are taken out
from the -80 oC freezer and quickly streaked onto LB agar plates containing appropriate
antibiotic for selection (eg, 30 g/mL of kanamycin for pET30 ek/LIC plasmids). They
are then incubated for overnight at 37 oC.
2. One colony from each streaked LB agar plates containing selective antibiotic is
inoculated in 3 mL LB medium (with the same antibiotic) for approximately three hours
until the optical density at 600 nm reaches 0.45.
3. This culture is then transferred to an Erlenmeyer flask containing 50 mL LB medium
with antibiotic and grown at 37 ºC with shaking at 250 rpm until the culture reached an
OD600 of 0.450.50.
4. IPTG is added to a final concentration of 1 mM to initiate the induction of protein
expression and the cells are then grown at 37 ºC for over night or 5 hours (to be
optimized based on particular clone). (Note: the volume of IPTG used should be adjusted
according to the amount of culture consumed for OD measurements).
Protein extraction.
5. After induction, each culture is harvested and resuspended in 500 L of 1X Protein
Extraction Reagent (10X Protein Extraction Reagent should be diluted to 1X using PBS).
6. Dilute Benzonase 10 fold with PBS, add 5 l of the diluted Benzonase into the cell
suspension (500 l). Higher Benzonase volume should be added if higher cell suspension
volume is used.
7. Add PMSF (a protease inhibitor) to a final concentration of 1 mM.
8. Incubate the cell suspension on a rotating mixer at a slow setting for 15 min at room
temperature.
9. Transfer contents of the Falcon tubes into labeled microfuge tubes
10. Remove insoluble cell debris by centrifugation at 13,000 rpm for 20 min at 4 °C (using
the bench top refrigerated microcentrifuge located in the Xu Lab).
11. Transfer the supernatant to a fresh tube and place on ice all the time.
SDS-PAGE analysis
12. To perform SDS-PAGE analysis, remove a small sample (15 l) and combine with 2X
SDS loading buffer (15l).
13. Heat the sample at 99 C for 5 min to denature proteins.
14. Centrifuge at room temperature for 1 min at 13,000 rpm.
15. Place sample on ice until all samples are ready to load.
16. Set up the precast gel onto the protein gel chamber and pour in 1X running buffer.
17. Load the samples (30 l) using protein gel tips (long tip) along with the protein marker (5
l).
18. Run at 90 volts for about 1 hour or until the dye tracer reaches near bottom of the gel.
19. Stain the gel in Biosafe Coomassie Blue solution with shaking (1 hr to overnight).
20. Destain in distilled water.
53
21. Take a digital picture of the gel and determine if there is a heavy band of protein
corresponding to your expected fusion protein size (your native target protein size plus
4.5 kDa).
Scale up cell growth and protein purification
22. If your target protein is over-expressed, you need to scale up the cell growth and
induction procedure to 50 ml of culture for the condition deemed appropriate (5 hr or
overnight induction).
23. Repeat steps 1 to 4 to obtain cell pellet.
24. Repeat steps 5 to 11 with scaled up (5 fold) volumes for each operation. More than 1
microfuge tubes can be used for each sample in repeating steps 9 – 11. The crude protein
extracts contain His-tagged protein are placed on ice before purification.
25. add 2 ml of sterile, DI water to the dry column and wait for water flowing through the
column
26. Gently shake to suspend the resin and add 0.5 mL of His-band resin (settled bed vol
would be 0.25 mL) to the purification chromatography column
27. Wash the column with the following solutions in sequence:
a. 3 vol of sterile DI water
b. 5 vol of 1X Charge Buffer
c. 3 vol of 1X Binding Buffer
28. Allow 1X Binding Buffer to drain to top of column bed
29. Load column with 1 mL crude protein extract, then stop the flow and gently mix by
pipetting and incubate for 10 minutes at 4 ºC.
30. Start the flow and collect the Flow-through in a tube labeled “Flow-through & Binding”.
31. Wash column with 10 vol 1X Binding Buffer
32. Wash column with 6 vol 1X Washing Buffer into the column and collect the flow
through in a tube labeled “Washing”.
33. Add 0.5 mL 1X Elute Buffer into the column and collect the flow through in a tube
labeled “Elute”.
34. Repeat steps 29-33 until all the crude protein extracts are treated.
35. Add 1 mL 1X Strip Buffer into the column and collect the flow through in a tube labeled
“Strip”.
36. Run a polyacrylamide gel electrophoresis for all tubes of protein preparation of different
treatments. The majority, if not all, of purified His-tagged protein should be observed
from “Elute” tube.
37. Purified protein is stored at 4 ºC or on ice for immediate use, or combined with equal
volume of 75% sterile glycerol and aliquoted and frozen at -80 ºC for long term
storage/use.
REFERENCES
Novogen Protocol TB054
Sambrook and Russel. 2001. Molecular Cloning. 3rd ed. Cold Spring Harbor Laboratory Press. Cold
Spring Harbor, NY.
54
REPORT AND QUESTIONS (Exercise 13)
Your name:______________________
A. Report
Describe your group’s result of protein over-expression and purification analysis using SDS-PAGE.
B. Answer the following questions:
1.
What is the property of the His Bind resin that allows binding to his-tagged proteins?
2.
Why would you expect to see a protein band slightly (4.5 kDa) larger than the calculated
mass of your target protein?
55
EXERCISE 14. Polyacrylamide gel electrophoresis
OBJECTIVE


Understand the principles of polyacrylamide gel electrophoresis
Become familiar with the operation of minigel apparatus and subsequent gel staining/destaining
INTRODUCTION
Most biochemistry and molecular biology research laboratories in academia and industry will need to
purify, identify and analyze proteins. For a target-based drug discovery process, purifying and analyzing
protein targets is critical. Analyzing complex protein extracts (determining over-expression of target
proteins) or purified proteins (whether or not a protein is pure) requires use of polyacrylamide gel
electrophoresis. Similar to agarose gel electrophoresis, polyacrylamide gel electrophoresis is based on
electrical current to separate macromolecules according to their sizes (smaller sized molecules move
faster). They differ in that polyacrylamide gel matrix provides better resolution than agarose gel. For
example, polyacrylamide gels can resolve single base difference of short nucleotide chains. For protein
analyses, the samples have to be denatured and coated with detergent by heating in the presence of
sodium dodecyl sulfate (SDS) and a reducing reagent. SDS coating gives the protein a high net negative
charge that is proportional to the length of the polypeptide chain. After the samples (and a known protein
ladder) are loaded on a polyacrylamide gel and high voltage is applied, proteins migrate toward the
positive electrode. Since all proteins have net negative charge that is in proportion to their sizes, the
proteins are separated solely based on their molecular mass. The molecular mass of a protein can be
estimated by comparing the gel mobilities of a band with protein standards. Following gel
electrophoresis, protein bands may be visualized by Coomassie Blue or silver staining, with the latter
having higher sensitivity.
For this exercise, precast polyacrylamide minigels will be used to save lab time for preparing gels
and minimize exposure to hazardous acrylamide solutions.
MATERIALS
Tris HEPES SDS Running buffer (Thermo Scientific #28398 )
Miniprotein gel apparatus (Biorad)
Perfect Protein Markers 10-225 kDa (Novagen #69079-3
Fisher Pierce Precast SDS-PAGE gel (Fisher PI25201)
Protein gel loading tips (Fisher #02707138)
Coomassie Blue (Gel Code Blue Safe; Fisher #24596)
Shaking
Heat block
2X loading buffer
PROCEDURE
1. Combine 15 l of protein sample with 15 l of 2X loading buffer.
2. Heat for 5 min at 95 °C heatblock and immediately place on ice.
3. Set up precast gel onto miniprotein gel apparatus; pay attention to the rubber liner
(orientation) to avoid leaking.
4. Add running buffer to inner tank to check for lack of leaking
5. Add running buffer to the outer tank (to the level of about 1/3 of the height of the tank).
56
6.
7.
8.
9.
Prerun the gel without sample for about 15 min.
Flush the wells with buffer using a P-200 pipet using the running buffer.
Load the samples (about 30 l) along with protein markers (5-7 l).
Run at 130 volts for about 45 min or until the dye trace migrates to near the bottom of the
gel (about 1 cm from the bottom).
10. Disassemble the gel apparatus and carefully dislodge the protein gel into a box contain DI
water.
11. Rinse the gel three times with DI water with shaking to remove SDS (5 min per rinse).
12. Stain the gel by adding Coomassie Blue to barely cover the gel; allow to stain with slow
shaking for a few hours to overnight.
13. Destain the gel with DI water for a few hours or several days with water change (cover
the box with parafilm to avoid water totally dry out).
14. Take a photo image of the gel using a digital camera.
REFERENCES
Promega Protocols and Applications Guide 1996
57
REPORT AND QUESTIONS (Exercise 14)
Your name:______________________
A. Report
Describe your group’s result of scaled-up protein over-expression and purification analysis using SDSPAGE (the gel picture needs labels).
B. Answer the following questions:
1.
What is the role of SDS during SDS-PAGE?
2.
If no precast gels are available, briefly describe what reagents are required to make a
polyacrylamide gel for SDS-PAGE (some research on your own).
3.
If the heatblock temperature reaches 99 °C, what would you expect with your protein gel
results?
58
EXERCISE 15. Protein dialysis and concentration determination
OBJECTIVE



Appreciate the requirement of removal of salts and other unwanted materials from the purified
protein
Understand the concept of dialysis and various dialysis strategies
Become familiar with use of dialysis tubing for removal of small molecules from purified
proteins
INTRODUCTION
After his-tag proteins are eluted from the His-Bind columns, the unwanted ingredient (imidazole) has to
be removed from the protein preparation to avoid its impact on subsequent assays. Similarly, when
proteins are purified via conventional means of ammonium sulfate precipitation and chromatography,
various salts used for elution have to be removed. Even for pure protein preparation but in an undesirable
buffer, there is a need for switching buffers. In these situations, a technique called dialysis is employed.
Dialysis is defined as separation of smaller molecules from larger molecules or of dissolved substances
from colloidal particles in a solution by selective diffusion through a semi-permeable membrane. The
most widely used and economic method of dialysis is by using dialysis tubing. Other variations include
Nanosep centrifugal tubes and Slide-A-Lyzer dialysis cassettes.
In this exercise for our purpose, we will use dialysis tubing to get rid of imidazole and Nanosep
tubes to concentrate the purified protein target FabI.
MATERIALS
Dialysis tubing (Fisher #211524)
Pall Nanosep centrifugal devices 3 K cut-off (Pall #OD003C33)
PBS buffer
PROCEDURE (PERIODS DEFINED)
1. Estimate the length of tubing based on the amount of protein. Cut the tubing for a little
longer (~2 inches) than your estimation.
2. Soak the tubing in DI water for about 20mins until it is loosened.
3. Fold one end of the tubing twice and seal with a clamp.
4. Fill the tubing with protein solution.
5. Fold the other end twice and seal with clamp provided (Wear gloves and handle with
care, tubing is very soft).
6. Attach a buoy to one of the clamps (if multiple protein samples need to be dialyzed, label
the clamps accordingly to avoid confusion).
7. Place the tubing containing protein solution in a beaker filled with 1X PBS/10% glycerol.
8. Put a stir bar in the beaker with proper size.
9. Dialyze in the cold room (4 ºC) on a magnetic plate with moderate stirring speed.
10. Change PBS after 2 hrs, then change PBS every 4 hrs or dialyze for overnight.
11. Collect the dialyzed protein solution with extra care.
12. Pre-rinse the Nanosep devices with 500 l of buffer to get rid of trace amounts of
glycerin and sodium azide (by centrifuging at 14,000 x g).
59
13. Place the protein solution into the Nanosep centrifugal device (500 l maximum per
device) and cap the device.
14. Place the devices into a fixed angle centrifuge rotor that accepts 1.5 ml tubes (Caution:
always counterbalance the rotor with another Nanosep device containing an equivalent
sample volume).
15. Spin up to 14,000 x g for between 5 min to 20 min (depending on the fold of
concentration and volume of the sample).
16. At the end of the spin, remove the Nanosep devices. Concentrated sample is recovered
with a micropipette.
17. Determine the concentration of protein using Nanodrop spectrometer.
18. Prepare aliquots of protein for storage in a freezer and use later.
60
REPORT AND QUESTIONS (Exercise 15)
Your name:______________________
A. Report
Include results of your protein concentration determination below using Nanodrop.
B. Answer the following questions:
1. Define dialysis.
2. If you don’t have dialysis tubing but only Nanosep, how would you dialyze the pure protein
solution containing high concentration of imidazole which will inhibit enzyme reaction later?
3. Search for Slide-A-Lyzer and briefly describe how it works.
61
EXERCISE 16. Enoyl acyl carrier protein reductase enzyme assay
OBJECTIVE


Understand the reaction catalyzed by FabI and principle of in vitro FabI enzyme assay
Become familiar with the enzyme assay in a microplate format
INTRODUCTION
Enoyl acyl carrier protein reductase (FabI) is an essential enzyme involved in the bacterial Type II fatty
acid biosynthesis (FASII). It catalyzes the reduction of enoyl group attached to an acyl carrier protein
(ACP) dependent of nicotinamide adenine dinucleotide (NADH) (see Reaction 1 below). In order to
determine the enzyme activity of FabI, crotonyl-coenzyme A (CoA) and NADH are used as the in vitro
substrates (Reaction 2). During the reaction, NADH (which has an optimum absorbance at 340 nm) is
converted to NAD+, thus permitting monitoring of the reaction via the concentration of NADH.
Specifically, the activity of FabI enzyme is correlated with the speed of NADH to NAD+ conversion, i.e.,
under appropriate conditions the faster the decrease in absorbance in 340 nm, the higher the enzyme
activity. In contrast, if the enzyme is inactive or is inhibited by an inhibitor, less or no decrease in the
absorbance should be observed. Consequently, this reaction can be used to screen for enzyme inhibitors
which potentially could be developed into new antibiotics.
Reaction 1:
I
Fab
trans-2-enoyl-ACP + NADH - H + - 2e
itor
hib
n
i
+
No reaction
Fa b
I
acyl-ACP + NAD+
Reaction 2:
Crotonyl-CoA + NADH  butyryl-CoA + NAD+
In this class, FabI was one of the original drug targets for one of the groups. Since the enzyme
assay has been developed in the Xu Lab and it is not trivial to develop other chosen drug targets (it may
take weeks and sometimes required radioactive substrates), FabI assay will be used by all groups to
experience development of an assay for high throughput screening.
MATERIALS AND EQUIPMENT
Purified FabI protein
Crotonyl-CoA (Sigma, Cat. #: 50-901-07190)
NADH disodium salt (Calbiochem, Cat. #: 481913)
Triclosan (Sigma, Cat. #: NC9805111)
Isoniazid (Sigma, Cat. #: AC12260-0050)
Triclosan
Multi-channel pipette (Fisher)
Corning 96-well half-area microplate (ThermoFisher)
Tecan Genios plate reader (Tecan)
62
Tomtec Quadra3 Liquid Handler (Tomtec)
Multidrop dispenser with RapidStak (ThermoFisher)
PROCEDURE
1.
2.
3.
4.
5.
6.
7.
8.
Prepare 2 fold serial dilutions (six dilutions) of a FabI inhibitor (isoniazid) from a stock solution
with appropriate diluent (each group performs this step).
Using a multi-channel pipet, add 5 l/well of the above 10 X concentration dilutions into the top
seven rows of a half-area 96-well microplate, with the last row containing only diluent of the
same volume.
Using a multi-channel pipet, add 40 l/well of 1.25X NADH/FabI/Buffer master mix into all
wells of the same microplate.
Read the background reading in a plate reader (located in the Xu Lab) at 340 nm; this is 0 min
reading.
Using a multi-channel pipet, add 5 l/well of the 1.1X crotonyl CoA solution (another substrate)
into all wells except H7 through H12 (these six will serves as NADH no change control), which
will received 5 l of DI water instead.
Read the plate at 10 min and 30 min intervals in the same plate reader.
The wells (H7-H12) with the no or little decrease in 340 nm absorbance will be used as NADH
control which is really the absolute 100% inhibition. Those wells without any drugs (H1-H6)
serve as 0% inhibition. We will normalize the readings, plot the inhibition percentages as the
function of drug concentration and perform non-linear curve regression using Prism software.
IC50 values can be obtained from Prism analysis.
REFERENCES
Ling et al., 2004. Identification and characterization of inhibitors of bacterial enoyl-acyl carrier protein
reductase. Antimicrobial Agents and Chemotherapy. 45:1541-1547.
1.25 X NADH/DTT/FabI/Buffer master mix
125 M NADH
1.25 mM DTT
1.25 g/ml FabI
125 mM Sodium Phosphate Buffer (pH 7.5)
63
REPORT AND QUESTIONS (Exercise 16)
(20 pts)
Your name:______________________
A. Reports
Describe the natural cellular reaction enoyl acyl carrier protein reductase catalyzes in bacterial cells (2
pts).
Explain the strategy used in this exercise to determine FabI’s enzyme activity (2 pts).
In our final FabI enzyme assay determining enzyme inhibition by triclosan, please draw a figure
illustrating the assay design involving various controls and inhibitor dilutions used, accompanied by the
kinetic reading profiles image of the microplate. Include a figure legend to explain assay design in various
wells properly. (a suggestion: assign assay design as Figure 1A and kinetic profiles as Figure 1B). (5 pts)
Each group was assigned to analyze the raw data to generate a dose response curve using a particular
endpoint. Please describe the dose response curve using a figure and a figure legend. (5 pts)
64
B. Answer the following questions: (2 pts each)
1. What is the optimum absorbance of NADH?
2. Which pathway is enoyl acyl carrier protein reductase involved?
3. Why was crotonyl CoA added last, instead of being integrated as a component of the master mix
together with NADH and FabI?
65
EXERCISE 17. High Throughput Screening – growth inhibitors
OBJECTIVE



Understand the principles of forward and reverse chemical genomics
Understand the practices of high throughput screening approaches and the operation of laboratory
robotics
Expose to use of laboratory robotics in drug discovery process.
INTRODUCTION
In the mid 1980s it was considered that the battle against infectious diseases was practically over. More
than 20 years later we find a dramatic increase in multi-drug resistant pathogenic strains, emerging with
more frequency.1,2 The emergence of these multi-drug resistant pathogens presents a serious threat to the
public health, underscoring the need for new antibiotics.3 Different approaches to the discovery of novel
antibiotics have been used: modification of known antibiotics and the discovery of molecules that are not
affected by the resistance.2 The latter can be accomplish by screening large libraries of compounds
against bacterial targets (cells, receptors, enzymes, proteins, etc); this is more commonly known as high
throughput screening (HTS).2 The rapid advances in microbial genomics and laboratory robotics have
increased the interests for antibacterial drug discovery focusing on large scale HTS for protein inhibitors.
In this exercise, we aim to identify antibacterial inhibitors from natural product compound libraries by
high throughput cell-based screens.
MATERIALS AND EQUIPMENT





















Corning 96-well Microplates (ThermoFisher)
Compound Plates with working concentrations of compounds
10 ml of Gentamycin (200 µg/ml)
Minocycline
10 ml of 10% DMSO Solution
Pipets (P1000) and pipet tips (sterile)
McFarland Tubes
Inoculating loop
Target organism to be screen against
1.1X Mueller Hinton II broth cation adjusted (MH)
Tripticase Soy Broth (TSB)
Tripticase Soy Agar plate with 5% sheep blood
Multichannel pipet (5-50µl) and pipet tips (sterile)
Quadra 3 Tips
Thermowell Sealing Tape (Aluminum) and roller
Gloves
70% Ethanol
ddH20
Tecan Genios plate reader (Tecan)
Tomtec Quadra3 Liquid Handler (Tomtec)
Multidrop dispenser with RapidStak (ThermoFisher)
66
PROCEDURE
Compound library replication and liquid handler
1. Thaw out a number of Working Compound Plates 1-2 hrs before the screening.
2. Place 10 ml of gentamicin (200 g/ml) on to reagent reservoir.
3. Using a multi-channel pipet, add 10 µl of gentaiycin onto Column 12 of each Assay Plate (Positive
Control).
4. Place 10 ml of 10% DMSO on to reagent reservoir.
5. Using a Multichannel pipet, add 10 µl of 10% DMSO onto Column 1 of each Assay Plate (Negative
Control; see Figure 1).
6. Clean all Quadra3 Towers with 70% Ethanol.
7. Once the Compound Plates are thawed, place an appropriate number of Assay Plates (2 plates for
every Working Compound Plate) on Tower A with no lids.
Figure 1. Plate Format
1
A
10
B
10
C
10
D
10
E
10
F
10
G
10
H
10
Growth
C
2
10
10
10
10
10
10
10
10
3
10
10
10
10
10
10
10
10
4
10
10
10
10
10
10
10
10
5
10
10
10
10
10
10
10
10
6
10
10
10
10
10
10
10
10
7
10
10
10
10
10
10
10
10
8
10
10
10
10
10
10
10
10
9
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
Unknown Compounds
11
10
10
10
10
10
10
10
10
12
10
10
10
10
10
10
10
10
20XMIC
ab
Note: All wells contain 10 ul of 10% DMSO or 10 ul of unknown compound/control antibiotics in 10% DMSO,
all compounds should be in 20 fold more concentrated than final expected concentrations.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Place Working Compound Plates on Tower B of the Quadra3.
Make sure A1 positions of plates correspond to A1 corners of towers.
Turn on the Quadra3 and the laptop computer
Open the program Quadra 3 Command v4.
File, Open, Click on HTS assay plate replication 10µl.qpg
Go to System, Click on Connect, once done, click on System, then Initialize.
Go to System, Click on Run Program and follow the instructions. Note: The program will ask you
to place 80 tips (Column 1 and 12 should be empty) Position 6.
To turn Program off go to Step 18: Quit program at Pos 5.
File Exit.
Take plates from Towers C and D.
Clean all Towers with 70% Ethanol.
Cover each Working Compound Plates (from Tower C) with Thermowell Sealing Tape (Aluminum)
using the roller followed by the lid and store them in the -80C freezer.
Preparation of inoculum and bulk dispensing
20. Streak a cell stock onto a blood agar plate the day before and grow overnight at 35C.
21. Inoculate 5-10 colonies into a 125 ml Erlenmeyer flask containing 20 ml sterile TSB.
22. Grow at 35°C with shaking at 200 rpm until it achieves an OD625 of 0.08 to 0.1.
67
23.
24.
25.
26.
27.
28.
29.
30.
Add 1.0 ml of bacterial culture into 5 ml of sterile TSB.
Adjust the turbidity of the culture with TSB to 0.5 McFarland units.
Load 1.0 ml of the above TSB into 50 ml MH
Turn on the Multidrop 384 and Rapid Stak.
Place Assay Plates from the previous section on stacker #1 without lids.
Make sure A1 positions of plates correspond to A1 corner of stacker.
Set the Multidrop to 90 µl, to 96 well plate setting, and to 12 columns.
Load the dispensing cassette as described in Figures A-D.
A
B
C
D
31.
32.
33.
34.
35.
36.
37.
Place tubes from dispensing cassette inside the Erlenmeyer flask containing inoculum.
Press Prime on Multidrop for 5 seconds.
Press Ready on Rapid Stack.
Press Run/Pause (►).
Once all Assay Plates are done press Restack.
Turn off Rapid Stack.
On Multidrop Press Empty.
68
38. Clean dispensing cassette by Pressing Prime with 20 ml of 70% Ethanol, and followed by 20 ml of
ddH20 .
39. Turn off Multidrop.
40. Remove Stacker from RapidStak and place lids back on.
41. Cover Assay Plates with lids.
42. Wrap stacked Assay Plates (4 at most) with plastic film to minimize evaporation.
43. Incubate plates at 35C for 16-18 hrs.
Reading results and Tecan Plate reader with Connect plate stackers
44. Take Assay Plates out from 35C and remove the lids.
45. Clean Towers with 70% Ethanol.
46. Place Assay Plates on Stacker Tower, and place on Input side.
47. Make sure A1 positions of plates correspond to A1 corner of tower.
48. Place other Tower on the Output side (must be empty).
49. Turn On Tecan GENios plate reader (button on the back).
50. Go to Magellan5 Software and Open it.
51. On Wizard List Window click Start Measurement, and then Click Next.
52. Look for method file EndpoingMultiplateWBarcodes, click Next.
53. In the Instrument Section, select Use Stacker.
54. Click Start.
55. Data would be automatically saved on
Documents&Settings\Allusers\Documents\Tecan\Magellan\wsp\barcodename?
56. File Exit Magellan 5.
57. Once all the plates have been read, remove plates from Tower.
58. Clean Towers with 70% Ethanol and place back.
REFERENCES
1. Xu, H. H., Real, L., 2006. Biochem Biophy Res Comm 349:1250-1257.
2. Donadio et al., 2002. J Biotech 99: 175-185.
3. Baniecki et al., 2007. Amer Soc Micr 51(2):716-723.
4. Quadra 3, Tomtec, Operation Manual, Rev. C 04/06
5. Rapid Stak 2x Setup Guide, Thermo Electron Corporation
6. Multidrop 384 User Manual, Thermo Electron Corporation, Rev. 3.1
7. Tecan Reference Manual for Magellan Rev. 2.0
Antibiotic Stock Preparation
Materials and Equipment:
Gentamycin (Sigma# G1264)
10% DMSO
0.2µm Sterile syringe filter (Fisher #09-719C)
10ml Syringe (Fisher #309604)
Weigh Boat (Fisher #02-204-1A)
Analytical Balance
Vortexer
Gentamycin Antibiotic Stock (200µg/mL)
Procedures:
69
1.
2.
3.
4.
5.
To determine the amount of the antibiotic powder:
Weight (mg) = Volume(mL) · Concentration (µg/mL)
Assay Potency (µg/mg)
To determine the amount of diluent:
Volume (mL) = Weight(mg) · Assay Potency (µg/mg)
Concentration (µg/mL)
Pour powder and diluent into a 15ml centrifuge tube. Completely dissolve powder by
vortexing tube.
Sterilize by membrane filtration and pour into a sterile 15ml centrifuge tube.
Store antibiotic stock at -20ºC and below.
Reagent Preparation
Materials and Equipment:
100% DMSO
Sterile Purified H20
250mL bottle (Fisher #06-414-1B)
50ml Serological Pipette (Fisher #0720017)
10ml Serological Pipette (Fisher #13-678-12E)
Pipette Aid
10% DMSO (200ml)
Procedures:
1.
In a sterile 250ml bottle combine 20ml of 100% DMSO and 180ml of sterile purified H20.
2.
Mix thoroughly, label, and store at room temperature.
Media Preparation
Materials and Equipment:
BBL Mueller Hinton II Broth-Cation Adjusted (BBL #212322)
BBL Trypticase Soy Broth (BBL #211768)
Purified H20
Diack Sterilization Monitor (VWR# 55710-400)
Autoclave Tape (Fisher # 11-880-10S)
Stir Bar
Weigh Boat (Fisher# 02-204-1C)
Spatula (Corning #3004)
8-500ml Bottles (Fisher #06-414-1C)
NaOH
HCl
Stir Plate
Weighing Scale
Beckman pH Meter
Autoclave
1.1x Mueller Hinton II Broth-Cation Adjusted (2 liters)
Procedures:
1.
Weigh 44g of dehydrated Mueller Hinton II-Cation Adjusted media.
2.
Pour powder into a 2 liter flask, add 1.8 liters of purified H20 and stir bar.
3.
Mix and warm gently allowing media to boil for 1 minute to completely dissolve the powder.
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4.
5.
6.
7.
Adjust pH to 7.3 ± 0.1 using NaOH or HCl.
Pour into 500ml bottles. Place autoclave tape on the outside of the bottle. Also make sure to
label with media name and date.
Insert sterilization monitor and place cap loosely onto bottle. Sterilization monitor will melt
when media reaches the appropriate temperature.
Place bottles into autoclave pan with approximately 3 inches of water. Autoclave at 116 to
121º for 15min.
Trypticase Soy Broth (2 liters)
Procedures:
1.
Weigh 60g of dehydrated trypticase soy broth media.
2.
Pour powder into a 2 liter flask; add 2 liters purified H20, and stir bar.
3.
Mix thoroughly and warm gently until powder is completely dissolved.
4.
Adjust pH to 7.3 ± 0.2, using NaOH or HCl.
5.
Pour into 500ml bottles. Place autoclave tape on the outside of the bottle. Also make sure to
label with media name and date.
6.
Insert sterilization monitor and place cap loosely onto bottle. Sterilization monitor will melt
when media reaches the appropriate temperature.
7.
Place bottles into autoclave pan with approximately 3 inches of water. Autoclave at 121º for
15min.
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REPORT AND QUESTIONS (Exercise 17)
(25 pts)
Your name:______________________
A. Reports
Name three robotic instruments used in HTS exercises and describe their respective roles (6 pts).
Describe the plate format used in growth inhibition screens (label growth control and positive control
wells (4 pts).
Each group was assigned to analyze the raw HTS data for several plates. List chemical structure of at
least 3 compounds identified to have at least 70% inhibition of bacterial cell growth (6 pts)
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B. Answer the following questions: (3 pts each)
1. What is the main difference between an enzyme inhibitor (such as a FabI inhibitor) and a growth
inhibitor?
2. Which approach does the growth inhibition screen in this exercise employ (circle one):
a. forward chemical genomics, or
b. reverse chemical genomics?
3. If time is not a limitation, what would you like to do next with the hit compounds we obtained
during this class?
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Appendix
Phosphate buffer saline preparation:
137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, adjust pH to 7.4 with
HCl/NaOH.
1.1 Media and Reagents Preparation
1.2 LB agar plates CM15 + glucose 1 Liter
LB Broth (Miller) (EM Science# 1.10285.5000)
Dextrose Anhydrous (EM Science# DX0145-1)
1.2.1.1 Agar (Difco #214510)
25g
2g
15g
1.2.1.2 Nano water to volume
Add LB, Dextrose & Agar to nano water and mix.
PH to 7.0 to 7.5
Autoclave
Suggest: Liquid media 500ml to 1 liter = 20 min. with bottles set in wire baskets in autoclave.
Media with agar 500 ml to 1 liter = 30 min. with flasks set in wire baskets in autoclave.
Media with agar over 1 liter in volume = 45 min. with flasks set in wire baskets in autoclave.
Cool the media: 45 to 48 degrees C.
Add 441.2 l Chloramphenicol 34 stock solution
Mix well and pour plates as needed.
1.3 LB Medium 1 Liter
LB Broth (Miller) (EM Science# 1.10285.5000)
25g
1.3.1.1 Nano water to volume
Add LB brother powder to nano water and mix.
PH to 7.0 to 7.5
Autoclave
Suggest: Liquid media 500ml to 1 liter = 20 min. with bottles set in wire baskets in autoclave.
Media with agar 500 ml to 1 liter = 30 min. with flasks set in wire baskets in autoclave.
Media with agar over 1 liter in volume = 45 min. with flasks set in wire baskets in autoclave.
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Caution: please do not use freshly prepared LB medium in assays and screens; fresh
medium turns brown very quickly with addition of MTS/PMS reagents, probably due to
lack of dissolved oxygen in the freshly autoclaved medium. We use medium at least two
days old.
1.4 1.1XLB Medium 900 ml final volume
LB Broth (Miller) (EM Science# 1.10285.5000)
25g
1.4.1.1 Nano water to volume
Add LB brother powder to nano water and mix.
PH to 7.0 to 7.5
Autoclave
Suggest: Liquid media 500ml to 1 liter = 20 min. with bottles set in wire baskets in autoclave.
Media with agar 500 ml to 1 liter = 30 min. with flasks set in wire baskets in autoclave.
Media with agar over 1 liter in volume = 45 min. with flasks set in wire baskets in autoclave.
Caution: please do not use freshly prepared LB medium in assays and screens; fresh
medium turns brown very quickly with addition of MTS/PMS reagents, probably due to
lack of dissolved oxygen in the freshly autoclaved medium. We use medium at least two
days old.
Chloramphenicol 10ml CM 34 Stock
1.4.1.2 Chloramphenicol (Fisher Biotech BP904-100)
0.34g
100% Ethanol -Dehydrated 200 proof
10ml
10cc syringe, syringe filter, 2ml sterile eppendorf tubes (5)
Note: Wear gloves and use aseptic technique in all steps.
Weigh out 0.34g antibiotic. Place in beaker with 5 to 8mls of Ethanol and a stir bar. Mix well.
Pour into a graduated cylinder. Rinse beaker with ETOH adding the liquid to the graduated
cylinder till a 10ml volume is reached. Cover the cylinder top with parafilm and invert till mixed.
Filter syringe sterilize into eppendorf tubes:
While still in package, twist syringe tip loose. Remove syringe from package and open filter package enough to allow syringe tip to enter.
Quickly screw syringe filter onto syringe. Hold syringe (with filter tip down) over open eppendorf tube, remove plunger, and fill to ~10cc mark
with antibiotic solution from graduated cylinder. Replace plunger and gently push down until you hear it “click”. Continue pushing till the 2 ml
line is reached, plus 2 more drops so a meniscus forms. Move syringe to next eppendorf tube. Repeat. Close eppendorf tube caps. Label and
store accordingly (freezer). Increasing the recipe accordingly will produce a larger batch.
1.5 DPBS 1 Liter
KCl
NaCl
KH2PO4
Na2HPO4
0.20g
8.00g
0.20g
1.15g
75
CaCl2.2H2O
133 mg
.
MgCl2 6H2O
100mg
Add nano water to volume.
Adjust pH to 7.35 using 1M HCl or 1M NaOH as necessary. Filter sterilize into aliquots.
4.0 M Xylose (Sigma # X-1500) 1 Liter
1.5.1.1 D (+) xylose
600.4 g
A 4M solution of D (+) Xylose (formula weight of 150.1) requires 600.4g brought up to 1L total
volume. Start with less than half the desired volume of liquid. Slowly add the compound while
stirring. Then bring up to desired total volume. To facilitate the compound into solution use a stir
plate with low heat. Bring to a temperature of 35-40C. Mix till dissolved.
Filter sterilize.
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