Mol Biol 3V03 COURSE ORGANIZATION 2014
The objective of this course is to familiarize the students with types of experiments and
techniques employed in microbial and molecular genetics.
Instructor: Dr.Turlough Finan
Office LSB 537. Tel: ext 22932. email: finan@mcmaster.ca
Via Avenue to Learn during the semester
Instructional Assistant:
Alison Cowie cowieal@mcmaster.ca
Teaching Assistants:
George diCenzo, Stephanie Jones and Andrew Duong
Technician:
Arlene Sutherland
Lecture/Lab Schedule: Term II 2013-14
Each week we will meet for a fifty minute lecture period (Monday, 9:30 A.M.-10:20 A.M. in
MDCL/1010) during which time we will review the upcoming laboratory sessions. In addition, I
will use this period to help you on the theory behind the laboratory exercises. Laboratory
sessions will be held on Tuesdays and Thursdays, 2:30 - 5:30pm, in LSB-102 and LSB-103.
Laboratory Working Groups and Work Organization
The laboratory periods will commence with a very short introductory talk. You will work in
groups of two students. Individual groups will be doing the same experiments. However, the
precise strains used by individual groups may differ.
The two afternoons per week allotted to this course are probably an underestimate of the
laboratory time required. Some of the experiments require that you to come to the laboratory for
brief periods outside of the two scheduled sessions. We ask the group members to divide these
responsibilities into fair shares.
There will also be pre-lab exercises/questions that must be completed before a new
section begins. These answers should be written in your lab notebook and their completion will
be marked under the “overall performance” section of your final mark. These exercises are
designed to help you when it comes time to write your lab report and to further understand the
procedures that are being used. You should read the entire section before doing the pre-lab
questions.
General Laboratory Course Outline:
The laboratory experiments are grouped into five sections. You are responsible for preparing a
laboratory report on each of the five sections. Below is a brief description of the material
covered in each section.
Section 1: Homologous and heterologous complementation of Escherichia coli and
Sinorhizobium meliloti mutants, and genetic linkage determined by transduction. You are given
two thi mutants, one dct mutant, and two plasmid clones; you have to assign the mutations into
complementation groups. You will transduce a Tn5-233 insertion into the S. meliloti thi
auxotrophs and a dctA mutant and screen for linkage of Tn5-233 to the above mutations.
Time: Approximately three weeks. These experiments can probably be done in four laboratory
sessions i.e. the mating of clones into the mutants, followed next day by streaking the mating
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mix to select transconjugants; then three or four days later screen for the unselected marker
(growth on succinate vs glucose).
Section 2: General transposon mutagenesis; Tn5-B20 mutagenesis of S. meliloti.
Isolation and identification of auxotrophs or carbon utilization mutants by replica plating. In
these experiments we would like the students to: A) determine the Tn5-B20 transposition
frequency per donor and per recipient. B) isolate one or two auxotrophs and determine their
auxotrophic requirements by the auxonography standard test procedure C) determine the linkage
of the auxotrophic mutation to Tn5-B20 (Nmr) and D) develop an estimation of whether the
mutagenesis was random.
Time: Four weeks.
Section 3: Linking phenotype (plasmid incompatibility) with genotype. You are given a plasmid
carrying a portion of the origin of replication from the S. meliloti pSymB megaplasmid. This
region causes the plasmid to be incompatible with pSymB. We will use random mutagenesis to
try and “knock out” the incompatibility phenotype and assess the nature of the mutations that
give rise to this knock out. We suspect that the nucleic acid region in question possesses a gene
and we will try to assess whether it is the expression of this gene which causes incompatibility.
Time: Total time 4 weeks.
Section 4: Gene cloning and expression. This section will include PCR amplification of a gene,
cloning of the amplified DNA fragment into an expression vector, confirmation of recombinant
plasmids through digestion by restriction enzymes, protein expression and analyses of protein(s)
on polyacrylamide gel.
Time: Three-four weeks.
Section 5: Expression of central carbon metabolism genes (Entner Doudoroff, gluconeogenesis
and TCA cycle) in S. meliloti. In these experiments we will use lacZ gene fusions to determine
expression differences of central carbon metabolism genes grown in different media. Also, PCK
enzyme activity will be determined using a coupled reaction.
Time: Three weeks. We will grow the cells, spin them down, prepare cell extracts; students will
determine the standard protein concentrations, and phosphoenolpyruvate carboxykinase
activities, and then assay β-galactosidase activity for central carbon metabolism genes.
Note: There will be overlap in the timing between the sections; multiple experiments from
different sections will be happening at the same time so taking good notes and labelling tubes
and plates properly is very important.
Evaluation:
Laboratory Reports..................
Quizzes……............................
Final Exam..........(April 1st) ........
Overall Performance................
approx. 50 %
approx. 25 %
approx. 20 %
approx. 5 %
The laboratory reports will be evaluated based on a marking scheme that will vary
slightly from one report to another. We offer the following scheme as a guide: Introduction (1),
Materials and Methods (1-2), Results (3), Discussion (3-4), Overall Report (1). There will be a
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penalty for reports which are handed in late; 10% one day late, 20% for two days late, 30% for
three days late, 50% for four days late and 100% for five days late.
The Final Exam will deal with both theory and practice of the material covered in the
lectures and laboratory exercises. Overall Performance will be evaluated from performance
during laboratory exercises and attendance.
In accordance with the "Senate Resolution on Course Outlines," the instructors reserve
the right to make changes in the course that may occur as the course progresses. In accordance
with University policy, academic dishonesty, including plagiarism, will not be tolerated. If you
need to clarify what constitutes plagiarism, please consult the statement on Academic Dishonesty
in the Senate Policy Statements available either in abbreviated form in the university calendar or
in entirety at: http://www.mcmaster.ca/univsec/policy/AcademicIntegrity2008.pdf.
The instructor and university reserve the right to modify elements of the course during
the term. The university may change the dates and deadlines for any or all courses in extreme
circumstances. If either type of modification becomes necessary, students will be given
reasonable notice with an explanation and an opportunity to comment on changes. It is the
responsibility of the student to check their McMaster email and course websites weekly during
the term and to note any changes.
Requests for Relief for Missed Academic Term Work
For absences from classes lasting up to 5 days: Undergraduate students may report absences
lasting up to 5 days by using the McMaster Student Absence Form (MSAF) on-line, selfreporting tool, in order to request relief for missed academic work. The submission of medical or
other types of supporting documentation is normally not required. Students may use this tool to
submit one request for relief of missed academic work per term. It is the prerogative of the
course instructor to determine the appropriate relief for missed term work in his/her course.
Immediately after using the online tool, students MUST contact their course instructor
regarding the nature of the relief. Failure to do so may negate the opportunity for relief.
For absences from classes lasting more than five days: Students who are absent more than
five days MUST report to their Faculty Office to discuss their situation and may be required to
provide appropriate supporting documentation.
For the reporting of more than one request for relief per term:
Students who wish to submit more than one request for relief of missed academic work per term
MUST report to their Faculty Office to discuss their situation and may be required to provide
appropriate supporting documentation.
The MSAF on-line, self-reporting tool cannot be used for any final examination or its
equivalent.
Some Important Dates: Classes begin: Monday January 6, 2013.
Last day for registration and adding or dropping courses: Tuesday, January 14.
Mid-term recess: Monday, February 17 to Saturday, February 22.
Last day for cancelling courses without failure by default: Friday, March 14.
Classes end: Wednesday April 9th.
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Detailed Molecular Biology 3V03 Laboratory Course Outline
Tuesday and Thursday afternoons are referred to as Day 1 and Day 2. Below, the
detailed schedule of laboratory exercises that we hope to follow has been outlined (Note
the actual schedule may change depending on progress of experiments. Due dates for
reports and dates of tests may be earlier or later than indicated - adequate warning will
be given if dates change).
Week 1; January 6th - 10th (CLASS Monday 6th)
Day 1. Meet in LSB-102 and 103. Transform the E. coli thi competent cells with pT8 and
pT9 plasmid DNA -- select for Tcr transformants (Exp.1). Competent cells and
plasmids will be provided. Discuss Course Outline.
Day 2. Streak E. coli transformant colonies onto M9gluc and M9gluc + Thi to test
for complementation.
Mate the plasmids pT8 and pT9 from E. coli MT607 into two S. meliloti thi
mutants and one S. meliloti dct mutant ---select for Smr Tcr transconjugants (Exp.
2).
Data Acquisition Exercise.
Transduce Gmr/Spr from RmF951 into the S. meliloti thi and dct mutants (Exp.3) --- i.e. you wish to determine if the Ω5079::Tn5-233 insertion in RmF951 is linked
to the various thi and dct mutations.
Streak out mating spots (Exp. 2) onto LB/Sm200Tc5
Week 2; January 13th- 17th (CLASS Monday 13th)
Day1. Screen the S. meliloti Smr Tcr transconjugants for complementation of the Thiand Dct- phenotypes by streaking on M9-glucose and M9-succinate (Exp. 2).
Remember to include your controls!
Start Section 2: Identification of mutations by screening for auxotrophs, start
Exp.4 (Tn5-B20 mutagenesis). Prepare the mating mixtures and spot onto LB
plates.
Day 2. Finish Section 1, Screen the S. meliloti Gmr/Spr transductants from Exp.3 for
Nm, Thi and Dct phenotypes (Exp. 3).
Resuspend the transposon mutagenesis mating mix and plate for viable counts
and insertion mutants. Bioinformatics Tutorial (Appendix I).
Week 3; January 20th- 24th (CLASS Monday 20th)
Day1. Record data from Section 1. Record data from Exp 4.
Start Exp.5. Patch 300 of the Tn5-B20 insertion colonies supplied onto minimal
media
Day 2. If possible, identify auxotrophs (Exp.5) from the screen plates and purify those
you find, using the master plates (LB/Sm200Nm200) as a source of cells (may wait
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till Week 4). Plate the supplied auxotrophs on minimal medium to isolate
revertants (Exp. 6).
Section 1 laboratory report due Tuesday Jan. 28th.
Quiz #1 Tuesday Jan. 28th.
Week 4; January 27th- January 31st
(CLASS Monday 27th)
Day 1 & 2. Identify auxotrophs (Exp. 5) from the screen plates and purify those you find
using the master plates (LB/Sm200Nm200) as a source of cells.
Examine plates for revertants, and screen revertants for antibiotic resistance
phenotype (Exp. 6 continued).
Start Section 3. Transform pTH1414 and pTH1415 into competent E. coli cells
Week 5; February 3rd - February 7th (CLASS Monday 3rd)
Day 1. Characterize by Auxonography, those mutants that we give to you, and those
auxotrophs you isolate (i.e. Exp. 5 continued).
Mate pTH1414 and control pOT1 plasmid into Rm5004, At123, and At125.
In lab lecture “the nature of the incompatibility sequence.”
Streak pTH1414 and pTH1415 transformants onto LBGm 10 plates.
Day 2. Serially dilute and plate mating suspensions onto selection plates. You should
also complete the screening of revertants for the relevant markers.
View the transformants under fluorimeter to determine if orientation matters with
respect to gfp expression.
Week 6; February 10th- 14th (CLASS Monday 10th)
Day 1. Test/Confirm the auxotrophic phenotype of your isolates.
Streak out At123, At123 (+ pOT1), At123 (+ pTH1414) transconjugants from
relevant selection plates.
Set up mating spots with Rm5004 and E. coli LE30 transformants.
Start Section 4. Set up PCR.
Day 2. Examine your auxotroph confirmation plates – you may have to come in on
Friday to collect final data.
Spread plate mating spots onto selective media (LB/Nm 200Gm60 for LE30
transconjugants; LB/Sm200Gm60 for DH5 transconjugants) to isolate mutants.
Analyze PCR product on Agarose gel and purify DNA from gel.
Quiz #2 Thursday February 13th.
Section 2 laboratory report due Thursday February 13th i.e. before break.
February 17th - February 21st- Mid-term Recess
Week 7; February 24th- February 28th (CLASS Monday 24th)
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Day 1. Examine your plates from the matings you did prior to the break.
Miniprep to isolate putatively mutant plasmid and send for sequencing to Mobix
Set up ligation between the ‘insert’ DNA fragment and vector.
Day 2. GFP analysis on A. tumefaciens strains with mutant plasmids and alignments of
mutants and other organisms in the computer lab
Transform ligation mixture in DH5α competent cells. You may have to come on
Friday to see your plates.
The TAs will inoculate single colonies from Section 4 in LB and appropriate antibiotic on
Monday for you to screen for positive recombinant plasmid at day 1 of week 9.
**During the break, the TAs will isolate incα mutants from S. meliloti, miniprep the S.
meliloti to obtain the mutant plasmids and then transform E. coli. Upon your return, you
will miniprep the E.coli inc mutants and send those plasmids for sequencing. The TAs
will also mate your mutant plasmids into A. tumefaciens. In addition to that, TAs will
restriction enzyme digest your gel-purified PCR products (‘insert’) and cloning vector
that you will use for ligation next week.
Week 8; March 3rd - March 7th (CLASS Monday 4th)
Day 1. Miniprep to isolate plasmids from cloning experiment and set up restriction
digest.
Day 2. Analyze the digested plasmids on agarose gel to confirm the positive clone.
Quiz #3, Thursday March 6th
Section 3 report due on Thursday, March 6th
Week 9; March10th- 14th (CLASS Monday 10th)
Day 1. Polyacrylamide gel electrophoresis to analyze proteins.
Day 2. Start Section 5: Standard Bradford assay
Week 10; March 17th- 21st (CLASS Monday 17th)
Day 1. Assay phosphoenolpyruvate carboxykinase activity in E. coli cell extracts.
Day 2. β-galactosidase assays of glycolysis and TCA cycle genes.
Week 11; March 24th – 28th
(CLASS Monday 25th)
Quiz # 4 Thursday March 27th
Week 12; March 31st – April 4th (CLASS Monday 1st)
Mol Biol Exam April 1st
Last Week of Laboratory Exercises. Complete Sections 4 and 5.
Reports on Sections 4 and 5 are due on Tuesday April 8th .
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