Transformation of E. Coli: Reestablishing the Lac Operon
Honors Biomedical Science 2
Redwood High School
2/16
¢ Background
B
acterial transformation is of central importance in molecular biology. It allows for the introduction and genomic
inclusion of genetically engineered or naturally occurring plasmids in bacterial cells. This makes possible the
propagation, genetic expression and isolation of genetically engineered bacterium. Much of the current research and
experimentation in molecular biology involves the transformation of E. coli.
T
he transformation process involves the uptake of exogenous DNA by cells, resulting in a newly acquired genetic
trait that is stable and heritable. Bacterial cells must be in a particular physiological state before they can be
transformed. This state is referred to as competency. Competency can occur naturally in certain bacterial species of
Bacillus when the levels of nutrients and oxygen are low. However E. coli does not enter a stage of competency unless
artificially induced. Competent E. coli cells are extremely fragile and must be treated carefully. Treatment to achieve
competency is not easy and demands a precise protocol involving chloride salts, such as calcium chloride, and sudden
hot and cold temperature changes. The metal ions and temperature changes affect the structure and permeability of the
cell wall and membrane, allowing DNA molecules to pass through. The exact molecular reasons why this occurs are
still not fully understood. Perhaps you will be the individual who solves this riddle. The answer to this mystery will
have enormous application in many different areas of biomedical science.
T
he strain of E. coli that will be used in this lab has been genetically engineered
so that the Z gene in its lac operon is not functional. This was achieved by
inserting foreign DNA into the middle of the Z gene. The tranformant in this lab is
a genetically altered plasmid called pGAL™, which carries a functional E. coli Z
gene. The goal of this lab is to transform the genetically engineered strain of E.coli
by inserting the pGAL™ plasmid into the host cell providing the call with all the
gene products of a fully functional lac operon.
T
he Z gene codes for the enzyme ß-galactosidase. This enzyme catalyzes the
breakdown of lactose (milk sugar) into its molecular monomers of glucose
and galactose. E. coli prefers to catabolize glucose. However, in a situation where
glucose concentration is low and lactose concentration is high (for example after
you have consumed a glass of milk or eaten a piece of cheese) the lac operon
becomes active causing the Z gene to produce the enzyme necessary to breakdown
lactose. Thus yielding the monosaccharide of choice: glucose.
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o test if the transformation has been successful you will use an altered form of lactose called X-GAL™.
ß-galactosidase recognizes X-GAL™ as lactose and cleaves the polymer into glucose and a form of galactose that
has a blue chromagen attached. As a result the colonies of successfully transformed E. coli should appear blue whereas
non-transformed colonies in the presence of X-GAL™ will appear white. Additionally, the pGAL™ plasmid also
contains a gene that codes for an enzyme called ß-lactamase which inactivates the antibiotic ampicillin. E. coli is not
naturally resistant to ampicillin. If the transformation is successful not only will you have reestablished the ability of
this E. coli strain to catabolize lactose but you (the mad scientist) will have genetically engineered this strain to be
resistant to ampicillin!
Ø Go directly to the head of the class and collect your drool powered holographic pocket protector.
O
ne part of the protocol instructs you to calculate transformation efficiency. It is both possible, and probable; that
less than 100% of the stock E.coli cells will be transformed. One confirmation of this occurrence is the presence of
white colonies growing on the medium. Consider the following:
HBM Challenge Question:
How is it possible that an un-transformed colony could grow on a medium that contains ampicillin?
¢ Protocol
Micro Test Tube Key
(to be kept in –20˚C cooler)
pGAL = pGAL Plasmid DNA; CB = Control Buffer; CaCl2 = 0.05M CaCl2 for competency
+DNA = Cells with pGAL; -DNA = Cells without pGAL for Control
1.
2.
3.
4.
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6.
Note: All pipet tips used are pre-sterilized and need to be treated as such.
Record initials on the + and - DNA tubes. Transfer 500 µl of CaCl2 into the -DNA tube.
Using a disposable bacteria transfer loop, transfer your assigned number of well-isolated colonies (1-1.5mm in
diameter) from a stock E. coli culture (Danger) – into the -DNA tube. With each transfer “twist/rotate” the loop in the
CaCl2solution ensuring all bacteria are transferred.
Vortex the -DNA tube until no clumps are visible and the suspension looks cloudy
Transfer 250 µl of the -DNA cell suspension into the +DNA tube.
Transfer 10 µl of pGAL into the +DNA tube. Flush. Transfer 10 µl of Control Buffer into the -DNA tube. Flush.
Quick Reference (positive control):
DNA and competent cells are combined in a 260µl suspension. After the cells have incubated with the DNA,
growth media (recovery broth) is added. Cells continue to grow through the recovery process, during which
time the cells repair their membranes, grow and express the genes of the newly acquired DNA.
Procedure For Transformation:
7. Cold-Incubate the cells in the +DNA and -DNA tubes at -20˚C for 10 minutes.
8. Immediately move the +DNA and -DNA tubes to a 42˚C water bath for 90 seconds.
6. Immediately move the +DNA and -DNA tubes back to the -20˚C cooler for 2 minutes.
7. Transfer 250 µl of Recovery Broth to both the +DNA and -DNA tubes. DO NOT let the tip touch the cell suspension in
either tube. Cap the tubes. Gently “flick and tap” each tube
8. Incubate the +DNA and -DNA tubes in a 37˚C water bath for 30 minutes.
9. After the recovery period, remove the tubes from the water bath and place them in your tube rack at your station.
Plating Of Bacterial Cells:
*agar composition is purposely not identified – based on lab results, you will be required to identify agar composition.
Depending on the plate, the agar was infused with either ampicillin and X-GAL, or just X-GAL
10. Secure 3 agar plates labeled A, B, and C. Label each plate with your initials. Additionally, label plate A Control 1; label
plate B Control 2 and label plate C DNA.
11. Use the following matrix for inoculating your plates one at a time. When inoculating the agar, transfer your assigned
volume (µl) of recovered cells to the top left of the prescribed plate. Immediately spread the cells with a sterilized
inoculating loop. Use the one-direction/90˚ technique. Cover the plate and allow the cell suspension to be absorbed (5 min).
TUBE
- DNA Tube
- DNA Tube
+ DNA
PLATE
Control 1 (A)
Control 2 (B)
DNA (C)
inoculate here
Spread cells in
one direction
Rotate plate: spread cells
90˚ to the first direction
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Resulting Streak
Pattern
Transformation Lab
Preparing Plates For Incubation:
12. Stack your group's set of plates on top of one another and tape them together. Make sure that each plate has your initials
on them.
13. After the cell suspension has been absorbed, invert the plates and place them in a 37˚C incubator overnight.
* for homework prepare a data table – see Lab Write-Up instructions on page 4
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Transformation Lab
Post Incubation Analysis
14. Data collection for each plate: create pictorials, count and record the number of colonies, and record detailed descriptions
¢ Lab Write-Up
The title of the lab, your name, date, and the following information is all you need.
1.
PROCEDURES: In paragraph form. Hint: you will be speaking directly to the effects of each step of the protocol in
the discussion section – as a result you need not explain the procedures here. (see Discussion section directions)
2.
DATA: Create data tables to reflect pictorials, number of colonies, and detailed descriptions for all three plates.
3.
CALCULATIONS: The amount of cells transformed per 1 µg of DNA is called the transformation efficiency. In
practice, as in this lab, very small amounts of DNA are used (5 to 100 nanograms) since too much DNA inhibits the
transformation process. In this protocol 50 ng (0.05 µg) of DNA was used per group. Begin by counting the number of
colonies on the plate labeled DNA (C). It is important to keep in mind that each colony originally grew from one
transformed cell. A convenient method to keep track of counted colonies is to mark the colony with a marking pen on the
bottom of the plate.
r Determine the transformation efficiency using the following formula:
Number of Transformants Final Volume at Recovery (ml)
x
= Number of Transformants/µg
µg of DNA used
Volume Plated (ml)
4.
DISCUSSION: Analyze and explain your results. Be thorough and use relevant references to your observations,
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information from your textbook, notes and the background information found in this lab. A portion of your discussion
section must refer to the individual steps of the protocol as they relate to the biochemical and physical changes occurring
during each step, and the importance of each step to the overall goal of the protocol.
Focus Question: How is it possible that an un-transformed colony could grow on a medium that contains ampicillin?
5.
CONCLUSION: (use your vast experience writing conclusion sections of lab reports to figure out what to write here)
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Transformation Lab