MICROBIOLOGY 480

MOLECULAR BIOLOGY OF MICROORGANISMS LABORATORY

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

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

Prerequisite:

Micr302 and one (or concurrent enrollment in one) of the following: Micr421, Micr425 or Micr460.

Required Text:

Lab Manual - Will be supplied in class.

Link to Dr. Clark's Section: html or pdf

Link to Photos of gels for 2005

Link to Dr. Bender's Section: html or pdf

Link to Dr. Haddock's Section: html or pdf (not yet available)

General Information

Instructors:
Dr. David Clark	Life Science III room 1010
Dr. Kelly Bender	Life Science II room 106
Dr. John Haddock	Life Science III room 1009
Teaching Assistants:
Holly Simons
Archana Panikkar
Rikhi Gon
Life Science II room 187
453-4797

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:

Exam	25
Complete Lab Report	25
Short Lab Report	15
Quizzes (3)	15
Laboratory Skills	20
Total Possible Points	100

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.  

Topics Covered

Basic Techniques

Genotypes and Phenotypes

Growth Rate and Cell Constituents

Assay of b-Galactosidase

Protein Polyacrylamide Gel Electrophoresis

Isolation of Total Genomic DNA

Agarose Gel Electrophoresis

PCR using Metabolic and Phylogenetic Primers

PCR of 16S rRNA Gene from a Pure Bacterial Culture

Cloning of 16S rDNA PCR Products

Preparation of Plasmid DNA

Non-Radioactive Labeled Probe Construction

Dot Blots & Colorimetric Detection

Phylogenetic Computer Analysis of 16S rRNA Sequences

DNA Sequencing and Sequence Analysis

Isolation of Genomic DNA for Cloning

Forensic DNA Analysis

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 &endash; 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:

Laboratory Report	Due Date	Points
Growth Rate & Cell Constituents	Sept 12	25 - complete
Protein Electrophoresis	Oct 3	15 - short
(including Protein Electrophoresis)
]PCR Amplification with Metabolic and Phylogenetic Primers	Oct 13	15 - short
(including Agarose Gel Electrophoresis)
Non-Radioactive Labeled Probe Construction, Dot Blots, and Colorimetric Detection	Nov 3	25 - complete
Sequencing and Sequence Analysis	Nov 11	15 - short
Biotechnology	Nov 18	25 - complete

Short laboratory reports (three 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 (three reports each worth 25 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:  2-4 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:  4-5 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 l10E6 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:

TABLE 4.	LDH Activity of Regulatory Mutants
	LDH (specific activity*)
	No buffer	HEPES	TAPS	AMPSO
STRAIN		pH8.0	pH8.5	pH9.5
NZN130 (Parent)	640	310	250	71
NZN131 (Mutant)	1690	850	1050	1050
NZN132 (Mutant)	690	780	710	190

*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:

% Gel	Bromo Phenol Blue	Xylene Cyanol
6	26	106
8	19	75
10	12	55

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.)

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.

Each week of classes has FOUR lab days.  Of these the ODD numbered days (Monday & Wednesday) are the formal lab periods and the EVEN numbered days are the intervening days (Tuesday and Thursday) when you may have to inoculate cultures, examine plates, etc.  [Days when nothing happens are included in case changes need to be made.] 

Week 1:              Basic Techniques  (BT) and Genotypes and Phenotypes (GP)

Day 1 LAB            Mon 22 Aug

            Dilutions, streaking and spreading (BT)

Day 2            Count colonies (BT)

Day 3 LAB            Wed 24 Aug

            Genotypes and phenotypes (GP)

Day 4            Record results (GP)

Week 2:              Growth Rate and Cell Constituents  (GRCC)

Day 5 LAB            Mon 29 Aug

            Growth rate determination by optical density and viable counts (GRCC)

Day 6            Store viable count plates in cold room (GRCC)

Day 7 LAB            Wed 31 Aug

            Chemical assays for protein and RNA (GRCC)

Week 3:             Assay of Beta-Galactosidase (BG)

Day 9 LAB            Mon 5 Sept = Labor Day - No Lab

Day 10             TAs will grow overnight cultures for beta-galactosidase (BG)

Day 11 LAB            Wed 7 Sept

            Induce lac operon and assay beta-galactosidase (BG)

Week 4:             Protein Polyacrylamide Gel Electrophoresis (PAGE)

Day 13 LAB            Mon 12 Sept

            Preparation of samples and run SDS-PAGE gels (PAGE)

Day 14            First destain (morning) & second destain (afternoon) (PAGE)

Day 15 LAB            Wed 14 Sept

            Observe gels and measure bands (PAGE)

Week 5:            First Exam

                        Isolation of Total Genomic DNA (IDNA)

Day 17 LAB            Mon 19 Sept                        First Exam

Day 19            LAB            Wed 21 Sept

                        Isolate total genomic DNA from two environmental samples (IDNA)

Week 6:           Agarose Gel Electrophoresis (AGE)

PCR using Metabolic and Phylogenetic Primers (MPPCR)

PCR of 16S rRNA Gene from a Pure Bacterial Culture (16SPCR)

Day 21            LAB            Mon 9/26

                        Agarose gel electrophoresis of DNA extractions (AGE)

                        Set up PCR reactions using metabolic/phylogenetic primers (MPPCR)

Day 22            TAs:  Refrigerate completed PCRs

Day 23            LAB            Wed 9/28

                        Agarose gel electrophoresis of PCRs and analysis of bacterial communities (MPPCR)

                        Set up PCR reactions of a bacterial culture using 16S rDNA primers; freeze the reactions (16SPCR)

Week 7:            Cloning of 16S rDNA PCR Products (C)

                        Preparation of Plasmid DNA (PP)

Day 25            LAB            Mon 10/3

                        TAs:  Place PCR reactions in thermal cycler at 8:00 a.m.

                        Agarose gel electrophoresis of 16S rDNA PCR reactions (16SPCR)

                        Ligation of 16S rDNA PCR products into TOPO TA cloning vector (C)

                        Transformation and plating of E. coli (C)

Day 26                        Pick two white colonies in SOB and grow overnight (PP)

Day 27            LAB            Wed 10/5

                        Plasmid preps and restriction digests of plasmid DNA (PP)

Week 8:             Non-Radioactive Labeled Probe Construction (PC) Dot Blots (DB)

Computer Analysis of 16S rDNA sequences (CA)

Day 29            LAB            Mon 10/10

                        Agarose gel electrophoresis of plasmid restriction digests (PP)

                        Set up PCR reactions for DIG-labeling of 16S rDNA (PC)

                        Prepare dot blots using environmental DNA from Day19 (DB)

Day 30             TAs:  Refrigerate completed PCR reactions

Day 31            LAB            Wed 10/12

                        Hybridize dot blots with DIG-labeled 16S rDNA probe (DB)

                        Computer analysis of 16S rDNA sequences (CA)

Week 9:            Colorimetric Detection of Dot Blots (DB), Second Exam

Day 33            LAB            Mon 10/17

                        Colorimetric detection of dot blots (DB)

Day 35 LAB            Wed 10/19                        Second Exam

Week 10:             DNA Sequencing and Sequence Analysis (DSSA)

Day 38LAB            Mon. 10/24

                        Plasmid prep., quantitation and purity

Day 40LAB            Wed. 10/26

                        AGE, submit plasmid for sequencing

Week 11: Isolation of Genomic DNA for Cloning (IGDC)

Day 43LAB            Mon. 10/31

                        Isolation of genomic DNA, conc./purity, restriction digestion

Day 44 Unscheduled Tues. 11/1

                        Store RD at 4^OC

Day 45LAB            Wed. 11/2

                        AGE, isolation from gel, conc./purity

Week 12: Biotechnology (B)

Day 48LAB            Mon. 11/7

                        Grow cells and induce expression of NDO

Day 50LAB            Wed. 11/9

                        Extract cultures, TLC

Day 52            Friday 11 Nov = Veterans Day Holiday

Week 13: Forensic DNA Analysis (FDA)

Day 53LAB            Mon. 11/14

                        Isolate genomic DNA, AGE, PCR

Day 55LAB            Wed. 11/16

                        AGE

Week 14: November 21-25 THANKSGIVING vacation

Week 15: Third Exam

Day 58 LAB            Wed. 11/30                        Third Exam

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