ASMCUE MEETING-2012
Lab Design Based on Scientific Methods
Madhusudan Choudhary, Sam Houston State University
mchoudhary@shsu.edu
Jorge L.M. Rodrigues, University of Texas-Arlington
Jorge@uta.edu
June 15 (Friday): 3:15 to 4:15 (San Mateo Marriott)
June 17 (Sunday): 9:45 to 10:45 (San Mateo Marriott)
Abstract: Lab Design Based on Scientific Method Enhances Undergraduate Research
Experience and Promotes Students Learning Skills.
Madhusudan Choudhary. Sam Houston State University, Huntsville, Texas
Undergraduate research experiences are pivotal for active learning and critical thinking. The
objective of this lab design was to introduce integrated molecular and bioinformatics approaches
in teaching that enhances students’ understanding about the basic gene structure-function,
promoter structure, and conservation of the protein structure-function. Two hypotheses were
investigated. First, an integrated scientific methodology promotes students’ understanding of the
genetic and genomic concepts as well as of skills to learn molecular and genomics methods.
Second, this new lab experience facilitates students’ transitions towards independent research
career goals. Amplification, DNA sequencing, and sequence analysis of ctrA gene (Cell-cycle
Transcriptional Regulator) from three species of Proteobacteria were carried out using the
combined approaches of molecular biology and bioinformatics. Our objective is to incorporate
research-based lab activities, which provide students first-hand research experience of the
standard scientific process. These activities include reading scientific literature, developing
hypotheses, designing experiments, making reliable and logical inferences, and preparing and
reviewing manuscripts. Students were assessed for 50 questions representing active,
collaborative and critical learning skills. The average test score of the class has significantly
increased from 26.31  4.27 (pre-lab) to 41.83  3.72 (post-lab); paired t-test, p < 0.05. Also,
scores in all three types of learning skills, active learning, collaborative learning, and critical
thinking have significantly increased after completion of the lab activities, which provided the
evidence for the learning outcome revealed by this study. Also, the number of students enrolled
in further independent research has significantly increased (2 = 15.081, p < 0.002), and a large
proportion students joining the independent research come from the group of students, who
completed this research-based lab activities. This newly designed research-based laboratory will
be shared with educators interested in adding this lab method into other biology courses.
Objectives:
1. Enhance students critical thinking, active and collaborative learning skills
2. Provide students with research experiences
3. Lean molecular and genomics methods
Background:
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It has been a long practice of predesigned undergraduate laboratory exercises in various
undergraduate courses across science departments. Students follow the steps from the lab manual
and do exactly what instructors planned for students to do in the laboratory. Students answer prelab and post-lab questions, and submit the lab report. Students learning outcomes were minimal
though these lab exercises, and students complain that the labs were boring and wasteful. We
recognize that the academic standard of the labs must be of high standard, which challenges
students to think critically, and should be reflective in their learning outcomes. Therefore, we
present here a lab design based on scientific method in order to promote students learning that is
complementary to the concepts thought in lectures, it must be developed by students own
research based experience.
Our goal in “Lab Design Based on Scientific Methods” is to present research based
approach where a lab will be carried out with scientific questions and will be investigated
following standard scientific methodology. Although, students will be provided a lab manual on
standard molecular methods, they are open to research of their own to follow other alternative
methods. We provided them a list of papers on Bacterial cell cycle and cell cycle transcriptional
regulator (CtrA) to read before lab session starts. On the first class day, students will discuss the
background information and propose three hypotheses and specific molecular and genomics
methods to investigate these hypotheses. Each group of students will conduct experiments and
keep record of the result in the notebook. On the last lab session, they will submit a draft paper
and provide peer-reviewed suggestions for further revision. Final paper will be submitted to the
instructor for grading. A rubric will be provided to students for grading.
Lab Activities:
Week 1: Paper discussion and proposal for hypotheses and methods
Week 2: Genomic DNA isolation and purification and primer design
Week 3: Polymerase chain reaction (PCR)
Week 4: Purification of PCR products and DNA sequencing
Week 5: DNA sequence analysis: Identification of ctrA gene and codon analysis
Week 6: Protein sequence analysis: amino acid composition analysis and motif structure analysis
Week 7: Promoter analysis and Transcription termination sequence
Week 8: Draft paper: Peer-review session
Project Background:
Caulobacter cresentus is a member of -Proteobacteria, and its genome has been completely
sequenced and annoated. CtrA is a cell-cycle transcriptional regulator, which controls ~25% of
genes, and it plays an important role to initiate the cell cycle. CtrA autoregulates its own
expression via a cell signaling mechanism, and the binding sites of CtrA are located in promoters
of many genes, including its own promoter. CtrA have been suspected to exist in proteobacterial species.
Hypotheses:
1. ctrA is present in all bacteria.
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2. CtrA sequences are highly conserved as they are essential in Proteobacteria.
3. As the expression of ctrA gene is cell-cycle stage dependent, ctrA expression is regulated by
common regulatory sequences in their promoters.
Experimental Design:
Hypothesis1: To test the first hypothesis, two strains of Rhodobacter sphaeroides,
Rhodopseudomonas palustris, and Escherichia. coli are used for genomic DNA isolation and
amplification of the ctrA gene.
Hypothesis 2: To test the second hypothesis, amplified ctrA product was purified and it was
subsequently sequenced. DNA and Protein sequences were analyzed using BLAST program.
Codon usage and amino acid composition in the ctrA gene were determined.
Hypothesis 3: To test the third hypothesis, promoter and transcription termination signal
sequences were analyzed.
Grading (100 points): Post-Lab exam (40 points), Lab report (50 points), and Lab Note book
(10 points)
Evaluation (40 points): Pre-lab and post lab exams were conducted for three learning areas,
active learning, collaborative learning, and critical thinking. Post-lab exam also constitutes the
part of the final grade.
Rubrics for the lab report (Total 50 points): Abstract (3 points), Introduction (9 points),
Methods (10 points), Results, figures and tables (10 points), Discussion (10 points), References
(4 points), Formatting and language (4 points)
Resources:
Websites:
•http://www.idtdna.com/SCITOOLS/Applications/PrimerQuest/Advanced.aspx
•http://www.ncbi.nlm.nih.gov/
Research Articles:
1. Altschul, S., W. Gish, W. Miller, and D. Lipman. 1990. Basic Local Alignment Search
Tool. Journal of
Molecular Biology 215: 403-410.
2. Chen, W. P., and T. T. Kuo. 1993. A simple and rapid method for the preparation of gramnegative
bacterial genomic DNA. Nucleic Acids Res. 21 (9): 2260
3. Dieffenbach, C.W., T. M. Lowe and G.S. Dveksler. 1993. General Concepts for PCR
Primer Design.
Genome Research 3 (3): 30-37
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4. Laub, M., C.L. Swaine, L. Shapiro, and H. McAdams, 2002. Genes Directly Controlled by
CtrA, a Master
Regulator of the Caulobacter. Cell Cycle PNAS 99 (7): 4632-4637
5. Monod, J. The Growth of Bacterial Cultures. Annu. Rev. Microbiol. (3): 371-394
6. Mullis, K. 1990. The Unusual Origin of the Polymerase Chain Reaction. Scientific
American, pp. 56-65.
7. Nakashima, H., K. Nilshikawa, and T. Ooi. 1986. The Folding Type of a Protein is Relevant
to the Amino
Acid Composition. J Biochem 99(1): 153-162
8. Sanger, F., S. Nicklen, A. R. Coulson. 1997. DNA sequencing with chain-terminating
inhibitors. Proc.
Natl. Acad. Sci. 74(12): 5463-5467
9. Stassen, M. L., Doherty, K. and Poe M. 2005. COURSE-Based Review and Assessment. OIR
Handbook
10. Suzek, B., M. Ermolaeva, M. Schreiber, S. Salzberg. 2001. A Probalistic Method for
Identifying Start
Codons in Bacterial Genomes. Bioinformatics 17(12): 1123-1130
Sample Questions
1. Reading background research papers helps _________
A. understand the topic of research
B. design the experiment
C. propose the tentative hypothesis
D. A and B
E. A, B, and C
2. Authentic background information can be collected from______
A. Research papers published in peer-reviewed journals
B. Publically available website
C. Textbook
D. Blogs
E. All of the above
3. What is a scientific hypothesis?
A. A question that is testable through experiment with proper controls
B. A big scientific question that can’t be tested
C. A scientific problem settled by scientific poll
D. A and B
E. A, B, and C
4. Isolation of genomic DNA is needed for
A. Cloning a gene
B. mRNA expression studies
C. Restriction fragment length polymorphism (RFLP) analysis
D. Amplifying by PCR
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E. All of the above
5. Food or drink is not allowed in the lab because of the food contamination
from sources like_____.
A. Contaminated bacterial culture
B. Unknown contamination from the previous lab
C. Other pathogenic bacteria in the lab
D. Mutagenic chemicals
E. All of the above
6. What is the purpose of adding “LYSIS SOLUTION” during the genomic DNA
isolation?
A. to break the bacterial cell wall
B. to break the cell membrane
C. to break the large circular DNA
D. A and B
E. All of the above
7.
Washing the DNA pellets with 70% ethanol
A. removes excessive DNA
B. removes excessive salt from the DNA
C. removes all RNA from the DNA pellet
D. removes left over proteins from the DNA pellets
E. None of the above
8.
Isopropanol is used to precipitate ________.
A. DNA
B. Protein
C. DNA and proteins
D. RNA
E. All of the above
9.
What are the features that are necessary for PCR primers?
A. Length
B. GC clamp at the ends
C. No secondary structure at the 3 ‘ end
D. Both primers should have about same melting temperature
E. All of the above
10. Polymerase chain reaction is used to amplify DNA and it requires the following
A. Primers and template DNA
B. Taq DNA polymerase
C. dNTPs
D. PCR reaction buffer
E. A, B, and C
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11. Heating cycle is required to
A. denature double stranded DNA
B. denature enzyme
C. denature a primer dimer
D. pair primers with template DNA
E. all of the above
12. If you get multiple nonspecific bands in the PCR product, how will you
modify your experimental protocol?
A. Lower the annealing temperature
B. Increase the annealing temperature
C. Lower the denaturing temperature
D. Increase the denaturing temperature
E. Lower the time for the extension step
13. Before a DNA sample is loaded in the gel’s well, it was mixed with the loading
Buffer, which helps sink DNA. This is due to the fact that the loading
buffer contains ________.
A. buffer
B. Glycerol
C. Blue dye
D. Tris
E. All of the above
14. To visualize DNA band in the gel, gel is stained with __________.
A. Ethidium bromide
B. Syber green
C. A and B
D. Loading buffer
E. All of the above
15. What is used to estimate the size of the DNA band of the PCR product?
A. Ethidium bromide
B. DNA Marker or ladder
C. Genomic DNA
D. Taq Polymerase
E. All of the above
16. Sanger’s sequencing reaction is different from normal PCR reaction as of following
A. Only one primer is used
B. Both primers are used
C. ddNTPs and dNTPs is used
D. Only dNTPs are used
E. A and C
17. Sanger sequencing reaction is based on chain termination chemistry, and this is
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due to the fact that some of the dNTPs contain_________.
A. dideoxy sugar
B. dideoxy nitrogenous bases
C. dideoxy phosphate
D. A and B
E. A, B, and C
18. At the end of the sequencing reaction, multiple DNA fragments are produced,
and these fragments are_____________.
A. precisely the length of DNA fragments of one nucleotide difference.
B. of distinct differnt lengths
C. of identical lengths
D. All of the above
E. None of the above
19. Which program is used to search the protein database using DNA sequence as a query?
A. BlastN
B. BlastP
C. BlastX
D. BlastT
E. All of the above
20. What % of nucleotide sequences have to be identical to predict that the given
sequence belongs to a particular individual strain?
A. 50%
B. 70%
C. 99%
D. all of the above
E. none of the above
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Abstract: Challenging undergraduates through a discovery-based laboratory course.
Jorge L.M. Rodrigues. University of Texas, Arlington, TX, 76019.
Microbiology undergraduate students take a number of laboratory courses to fulfill program
requirements. Traditionally, laboratory courses are structured around basic exercises, followed
by a questionnaire that will be answered at the end of the class period. Students describe these
exercises as easy, repetitive, and tedious. More importantly, learned skills are lost as students
progress towards the degree. I hypothesize that students will learn problem-solving skills when
properly challenged in class. In order to develop skills in classical and molecular microbiology
methods, I developed an upper division laboratory course, in which students are challenged to
isolate and characterize an unknown microorganism. Samples come from the student’s interest in
a particular environment. In the first teaching module, students receive general laboratory
protocols and are required to maintain laboratory notebook for the experiments. The American
Society for Microbiology instructions to authors serves as a guide for a mid-term and final
laboratory reports. During the second teaching module, students receive a number of tasks for
molecular characterization of the microorganism, but no protocols are provided. Students are
challenged to identify research objectives through searches and discussion the current literature.
They develop their own experimental protocols for polymerase chain reaction, primer design,
and molecular taxonomy. Each student successfully characterized its isolate. Students enhanced
their quantitative skills through growth curve analysis and statistical significance of results,
gained laboratory skills for DNA amplification, cloning, and transformation, and learned to write
and evaluate technical literature.
Description of Course Description: This course is intended to provide students with an
overview of different techniques used during manipulation of microorganisms. It will allow
students to gain a historical perspective of techniques used in microbiology (Winogradsky
column, Koch solid agar plating) as well as learn state of the art molecular characterization of
microorganisms and their genetic manipulation.
Student course outcomes: This course is designed to provide undergraduates with the basic
information about molecular methods currently used in modern microbiology. At the end of the
course, undergraduates must be able to isolate and characterize microorganisms, to design an
experiment, to report its findings. They are expected to know and perform genomic DNA
isolation, polymerase chain reaction, cloning and transformation, and DNA sequence analysis.
Grades:
25% from Report 1
35% from Report 2
10% Quizzes
30 % from Laboratory notebook and Participation.
Laboratory Activities:
Week 1:
Week 2:
Week 3:
Week 4:
Making media (discussion on nutrients, pH, sterilization procedures)
Inoculation, Winogradsky column (discussion on dilution, enrichment, buffers)
Isolation (discussion on methods of isolation, Is there a need for isolation?)
Description (discussion on microscopy)
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Week 5:
Week 6:
Week 7:
Week 8:
Week 9:
Week 10:
Week 11:
Week 12:
Week 13:
Week 14:
Cell growth (exercise on growth rate)
DNA extraction (discussion on DNA extraction methods)
PCR (discussion on polymerases)
Gel electrophoresis (discussion on DNA separation, DNA properties)
Cloning and Transformation (Lac Z selection)
Plasmid extraction and gel electrophoresis (discussion on plasmid-mediated
resistance)
Total soil DNA extraction (Metagenomics)
Sequencing (Sequencing methods, visit to Genome facility)
Sequence analysis (databases)
Phylogenetic tree (discussion on relationships)
Book suggestion for this course (No, I do not make any money on this):
I Microbiologist: A discovery-based course in Microbial Ecology and Molecular Evolution. Erin
R. Sanders and Jeffrey H. Miller. ASM Press, ISBN 978-1-55581-470-0
Example of a lab handout provided to students:
BIOL 3445 - Methods in Molecular Microbiology
Lecture 05
7) Determination of a growth curve
Materials:
- 2 glass cuvettes
- Spectrophotometer
- Kim wipes
- P-1000 Micropipette
- P-1000 micropipette tips
- Shake incubator (37⁰C)
- 500 mL Erlenmeyer flasks
- Growth media
- 15 mL Falcon plastic test tube
- Disposable loop
- Aluminum foil
- Scotch tape
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Method:
Preparation:
1) Prepare three 500 mL Erlenmeyer flasks each containing 200 mL of liquid growth media
of your choice. See lecture 01 for protocol.
2) Select colony on which to determine the growth curve of and use a loop to touch a single
colony and inoculate one flask by placing the loop inside the liquid media and stirring it
vigorously.
3) Cover the top of the flask with aluminum foil and label it with the date and initials on a
piece of tape, then place the flask in 37⁰C for 2-3 days.
4) Place the 2 other uninoculated flasks in 4⁰C.
5) After 2-3 days, take the inoculated flask out of 37⁰C and check for growth. To check for
growth swirl the flask and look for sediment particles. If growth is observed then transfer
100 ul of media to one of the uninoculated flasks previously stored at 4⁰C (ensure there
has been no contamination) and allow it to incubate at 37⁰C overnight.
Determining a growth curve with a spectrophotometer:
6) To begin taking growth curve readings, a blank must be kept to zero the
spectrophotometer each time a measurement is taken. Therefore, take 8 mL of
uninoculated medium from the remaining flask kept at 4⁰C and transfer it to a 15 mL
falcon test tube.
7) Turn on spectrophotometer by turning left hand knob and allow it to warm up for at least
15 minutes, and adjust the wavelength to 600 nanometers.
8) Transfer 10 mL (or 5 % of total volume) of media from the flask inoculated overnight to
the uninoculated flask and swirl to mix.
9) Take 2 clean glass cuvettes and add 5 mL of uninoculated media from the 15 mL glass
test tube to one (blank), and 5 mL of media from step 8 to the other.
10) Carefully place the “blank” into the sample holder and close the lid and adjust the
absorbance value to “0”. (Note: Hold the cuvette by the upper rim and make sure to wipe
the outside of the cuvette with a kim wipe each time before placing inside the sample
holder to ensure that there are no fingerprints and that the cuvette is dry!)
11) Remove the blank and place the cuvette with the growth sample into the sample holder.
Close the lid and record the absorbance measured. Remove the dump the sample into a
waste beaker.
12) Take the flask from step 8 and incubate it in a shake incubator (37⁰C) for 30 minutes
before taking another reading on the spectrophotometer.
(Note: Remember to blank the spectrophotometer each time before reading the sample.)
13) Place the flask back into the shake incubator and continue to take readings every 30
minutes for several hours (5-10 hours).
(Note: A growth curve is complete once it has gone through a lag phase, exponential
phase, and stationary phase. When the absorbance reaches its maximum and remains
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relatively constant, then it has reached the stationary phase. If the generation time of the
microorganism is long, then it may take several days to complete a growth curve, in
which case, readings can be taken at 12 hour intervals rather than every 30 minutes.)
14) Once all readings have been taken, using Excel graph the absorbance measurements on
Y-axis against time on the x-axis.
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