Biology

Microbe Identification Paper

Outline

Rough draft is due at the end of Thursday, Nov. 15. Final draft is due at the end of Tuesday, Nov. 27. Remember, each student writes their own paper in their own words. The sooner I get your rough draft, the sooner you get it back.

I. Introduction (3 paragraphs – choose 2 of the first 5 and do number 6)

1) Bird feathers and bacteria

· Describe what bacteria are

· What types of bacteria may be found in bird feathers?

· What are they doing there: harmful, helpful, neutral?

2) You have sequenced the 16S rRNA gene in your microbial sample

· What is 16S rRNA

· What is this gene especially useful when determining phylogenetic relationships?

3) Phylogeny

· What is phylogeny?

· Describe why is it useful to know?

4) Sequencing

· What is Sanger sequencing?

· How has sequencing technique changed over the years

· Why is sequencing a valuable tool in molecular biology

5) PCR and gel electrophoresis

· What is PCR and gel electrophoresis?

· Describe two reasons scientists do PCR?

6) Hypotheses

· State the objective

· Predict which one of the sequences you collected from NCBI will be the closest match to the DNA sample you sequenced. Provide a reason for your prediction.

II. Methods (3 short paragraphs)

· Describe methods for PCR, gel electrophoresis, & sequence analysis.

III. Results and Discussion: (2-4 paragraphs)

· Include a labeled figure of your gel (I will send to you)

· Include a labeled figure of your phylogenetic tree

· Attach a “.masx” file showing your DNA sequence alignment

· Describe the results of your figures

· Interpret (discuss) the results, such as

· Did your PCR work properly?

· Which organism is most closely related to your sequence and is your hypothesis correct?

· Briefly describe the species or genus you found. Is there anything interesting about it? Is it surprising to find it on a bird?

· Name and describe one or two ways the design of this experiment could be improved?

IV. References

· Include at least one reference here

Example figure:

Figure 1. Growth of bacterial colonies that were irradiated for 2 minutes under UV light.

The figure should be easy to see and have a descriptive footer beneath. For your gel picture, you’ll want to label the lanes.

If you present a table, then it will have a header describing the table and it is placed above the table.

GRADING RUBRIC for FINAL PAPER

INTRODUCTION of 64 pts

– Is the general topic well described? of 9

– Is the relevant information included? of 9

– Is the information accurate? of 9

– Is information well organized and in a logical order? of 9

– Is there a clearly written hypothesis? of 10

– Is communication clear and effective? of 9

– Is plagiarism avoided? of 9

METHODS of 35 pts

– Are the relevant procedures included? of 7

– Are the methods accurate? of 7

– Is proper science terminology and grammar used? of 7

– Is plagiarism avoided? of 7

– Is information well organized and clear? of 7

RESULTS and DISCUSSION of 66 pts

– Are the PCR results presented in a well-captioned figure? of 6

– Are phylogeny results present in a well-captioned figure? of 6

 Is the ‘.masx’ sequence file attached and sequences aligned? of 6

– Are the results presented in the text? of 6

– Are the results properly interpreted? of 12

– Is the original hypothesis correct? of 6

– Is the unknown bacterium identified and described? of 6

– Is it recognized when the experiment went wrong

and does the student have a remedy? of 6

– Is communication clear and effective? of 6

– Is plagiarism avoided? of 6

REFERENCES of 15 pts

– Is at least one reference used and is format ok? of 5

– Is the reference shown in parenthesis where mentioned in text? of 5

– Is the reference accurate and properly used? of 5

An ideal paper is concise and contains the relevant information. No minimum or maximum length. Ask yourself if you’ve fulfilled the outline and grading rubric requirements.

TOTAL SCORE: _____ of 180 for final draft. (note: 20 pts for rough draft)

Methods:

Should include:

(1) Sample collection and DNA extraction (given below)

(2) PCR mix (what was is in the mastermix and what did you add to the mastermix?)

(3) PCR conditions (given below)

(3) PCR product cleanup (given below)

(5) Sequence analysis with Mega (this may be hard, but try your best to write this in a short paragraph)

Sample collection and DNA extraction: Birds were captured in the wild and swabbed on site to collect microbes, which were grown in nutrient broth before extracting DNA

PCR conditions: The PCR profile consisted of 96oC for 5 min, followed by 30 cycles of 94oC for 30 sec, 60oC for 90 sec, and 72oC for 60 sec. Amplification products were separated on a 1% agarose gel containing ethidium bromide and visualized under UV light.

Excess primers and nucleotides were removed from the PCR product by adding 4 uL ExoSAPIt and incubating for 15 mins at 37oC before deactivating the enzymes at 15 min at 80oC. Purified PCR products were sent out for Sanger sequencing.

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