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MICROBIOLOGY 480

MOLECULAR BIOLOGY OF MICROORGANISMS LABORATORY

Taught each Fall semester, 2:00-6:00pm, M W

Course Description:

Genetic and biochemical analyses of microorganisms using a variety of techniques in molecular biology, molecular genetics and biotechnology.

Prerequisite:

MICR 302 and one (or concurrent enrollment in one) of the following: MICR 421, MICR 425 or MICR 460.

Link to Syllabus and Lab Rules

Required Text:

Lab Manual - Will be supplied in class.

Link to Dr. Fix's Section: pdf

Link to Dr. Bender's Section (to be updated soon): pdf

Link to Dr. Haddock's Section: pdf

General Information

Grading:

Each of the three instructors will be in charge of one third of the course. There will be 100 points for each third of the course, awarded as follows:

Part of your grade for the course will be the instructors' assessment of your lab skills.  This will not be based on attendance (since you are expected to attend every lab), but on how well you follow the directions given to you, how you work with others in a group setting, and how well you take responsibility for the materials and equipment you are using in class. Please note that you will not be penalized in this area if one of your experiments fails. How you conduct the experiment is more important than the outcome.

Unlike lectures, lab courses involve a great deal of planning and preparation and also consume expensive materials. There will be no make-ups for any labs, exams or reports. Any unexcused absences will result in deducted points.

You must provide yourself with a lab coat and with marker pens that write on glass and plastic (e.g. Sharpie brand).

Topics Covered

Laboratory Reports

As indicated in the scoring for the course, your performance on the laboratory reports constitutes more than 40% of your grade. Laboratory reports are considered individual projects; that is, although you will be working in groups during lab, you are expected to write your own lab report.

All lab reports must be type written single-spaced in 12 point font. Graphs and tables may be hand-drawn. Reports must be received by no later than 5:30 p.m. on the due date. Late reports will be assessed a point penalty; 5 points will be deducted from the score for every day past the deadline.

Laboratory reports are due on the following dates and worth the points listed:

Short laboratory reports (two reports each worth 15 points; 10 points for results, 5 points for discussion) are to include only the results and an abbreviated discussion, which should be no more than one page.

Complete laboratory reports (two reports each worth 25 points and one report worth 40 points) will be formatted as in standard scientific publications (see Instructions to Authors in the January issue of the Journal of Bacteriology) and will include the following sections:

Introduction: Two-four concise paragraphs that describe the experiment and its objectives (i.e. the questions you are asking, the hypotheses you are proposing before you actually start the experimental work). Not to exceed one page.

Materials and Methods: Include any additions to or changes from the procedure in the Lab Manual. Apart from this just say: "Please refer to MICR 480 Laboratory Manual".

Results: Four-five paragraphs describing the results (either for your group only or for the entire class, depending on the experiment). Where possible, results should be summarized in tables or graphs. You should number and provide a title for each table or graph. Refer to them by their number in the text portion of this section. Provide a legend for each figure describing what the data refer to and a description of the meaning of any symbols or abbreviations used. Footnotes may be used below tables.  Graphs should be constructed by hand plotting the data points. Computer-generated graphs are NOT acceptable.

Discussion: Discuss the principles underlying the experimental approach.  Explain your results with reference to these principles and any hypotheses you proposed in your Introduction. The Discussion should also include possible explanations for any problems you encountered, e.g. if you got only negative results. Not to exceed 2 pages.

Conclusion or Summary: A single paragraph summarizing your results and indicating whether or not the experimental approach was appropriate to test your hypothesis. Summaries of scientific papers are often distributed without the rest of the paper. The reader should therefore be able to understand your summary on its own.

Example Summary:

Under anaerobic conditions Escherichia coli converts pyruvate to D-lactate by an NADH-linked lactate dehydrogenase (LDH). The ldhA gene, encoding this LDH was cloned using λ10E6 of Kohara's collection as the source of DNA. The ldhA gene was sequenced. The ldhA gene of E. coli was highly homologous to genes for other D-lactate specific dehydrogenases but unrelated to those for L-lactate specific enzymes. We disrupted the ldhA gene by inserting a kanamycin resistance cassette into the unique KpnI site within the coding region. When transferred to the chromosome, the ldhA::Kan construct abolished the synthesis of the D-LDH completely. When present in high copy, the ldhA gene was greatly overexpressed, suggesting escape from negative regulation.

Example Table:

*Specific activity in nmoles of NADH min^-1 mg^-1 protein.

All cultures were grown anaerobically in rich broth plus 0.4% glucose for 6 hours at 37°C.

Buffers were used at 100mM.

Units & Conversion Factors etc

Metric Prefixes

M = mega = 10⁶

k = kilo = 10³

m = milli = 10^-3

µ = micro = 10^-6

n = nano = 10^-9

p = pico = 10^-12

f = femto = 10^-15

a = atto = 10^-18

Conversions

1 µg of a 1 kb DNA fragment = 1.5 pmol; 3.0 pmol ends

1 pmol of a 1 kb DNA fragment = 0.66 µg

10 kDa protein ≈ 270 bp DNA

1 kb dsDNA = 6.6 x 10⁵ Da

Concentration of nucleic acid (µg/ml) for 1 A₂₆₀ unit

dsDNA = 50

ssDNA = 33

ssRNA = 40

Oligonucleotides = 20

Dye migration in polyacrylamide denaturing gels

Size of DNA fragments (in bp) with which the dyes would migrate:

Laboratory Rules

Safety is our main concern in the lab. Every student must wear a lab coat at all times. Always follow directions carefully and especially be aware of the CAUTION notes in your lab manual. You will be told in your lab manual to wear gloves and/or eye goggles during certain procedures -- do so. There is to be absolutely no eating, drinking, gum chewing, or applying of cosmetics in the lab at any time. Anyone not following these directions will be asked to leave immediately -- there are no exceptions.

One of the instructors will be present during the first part of each lab period to explain what is to be done that day, discuss concepts, and answer any questions or problems. We may then leave the TAs in charge. You will always be able to find us during lab period either in our offices or labs. If we must be out of the building for a short time, we will announce it in lab. Please feel free to come and talk to us, especially if you are experiencing difficulties during the lab period (when you aren't actually in the middle of an experiment, of course). Remember, we will not know that there is a problem and will be unable to address it unless you inform us. We also welcome well-thought-out suggestions or comments on the lab, or any discussions pertaining to the concepts covered in the lab manual.

When we are not in the room, the TAs have our authority. This means that you will follow their directions and/or requests. You are not to argue with the TAs during lab period. If there is a problem, you will follow the directions the TA gives you and then discuss it with the TA and ourselves later. This rule is primarily a safety precaution (there may be something inherently dangerous in the way you are doing something) but is also a way to ensure that the lab runs smoothly for everyone. Cooperation is key.

As mentioned above, how you conduct the experiment is more important than the outcome. Keep this in mind during those times (and there are bound to be a few) when things don't seem to be working. If you conscientiously follow directions and think about what you are doing (both before and during the experiment), you should master those skills expected of you in this lab. But if you're sure you did everything right and your experiment still doesn't work, don't despair. There is such a thing as the fickleness of lab experiments and it happens at some time to everyone. That is why you are graded on your effort and not necessarily on your outcome. (If, however, none of your experiments work, some reexamination of your lab technique may be in order.)

Schedule of Experiments

General Comments

You will be required to come in at non-scheduled lab times to perform various chores such as taking your plates out of the incubator or putting your overnight cultures in the cold room. If your experiment is ruined because you forgot to come in and your colonies overgrew, you may lose points, so don't forget.

Keep good records of all of your results, including Klett readings, plate counts, etc. Also note any unusual results in your manual (for example, if your culture seems to take much longer than anyone else's to double). Many experiments rely on seemingly trivial details.

All dilutions are to be done in sterile saline unless otherwise instructed. Incubate all liquid cultures in a shaking water bath and incubate plates upside down in the incubator. Centrifugation is to be done only by the TAs. On a regular basis remove and discard plates or cultures in the cold room that you no longer need.

Detailed Timetable

Each week of classes has FOUR lab days. Of these, Monday and Wednesday are the formal lab periods and Tuesday and Thursday are the intervening days when you may have to inoculate cultures, examine plates, etc.

SCHEDULE

Related Microbiology Courses:

[ MICR 302 ] [ MICR 421 ] [ MICR 425 ] [ MICR 460 ]

SIUC / College of Science / Microbiology / micr480/
http://www.micro.siu.edu/micr480/index.html
Last updated: 08-Aug-11 / df