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Identification of Unknown: e. coli

Unknowns Experiment:
Each student was given an unknown bacteria. Over the next several labs, students will perform gram stains and biochemical tests to identify their unknown. In a lab near the end of the semester, we will do colony PCR on your unknown, amplifying the 16SrRNA gene. An aliquot of this 50 L PCR reaction (10 L) will be checked for a ~1.5 kb product using agarose gel electrophoresis, and if successful, the remaining 40 L will be purified and sent for sequencing. The returned sequence will be used to search the NCBI nucleotide database to find a closely matching bacterium that will (hopefully) support your biochemical analysis.

Tests Done:
Gram stain. Result: negative
Sucrose Fermentation: positive
Indol test: positive
Urease: negative
Conclusion: Unknown bacteria is e.coli.
Colony PCR has not been done yet. Parts of the Results and Discussions will be incomplete, but I can finish them later.

Report Instructions:
Title – make up one and include your unknown’s # (Unknown: 1)

Final reports should be written in scientific paper format as described in the handout.

Abstract – summarize the project, results and conclusions in one short paragraph.

Introduction – Present why identification of unknown bacteria is important in both environmental and medical microbiology, and why biochemical differences might occur among bacteria. Talk about molecular methods for bacterial identification, and the choice of 16S rRNA as a marker and why it is better than some enzyme to identify microbes. Give a background on what the 16S rRNA is, where its found, why it mutates less than most other genes, and tell about conserved vs. variable regions and what they can be used for. Finally end with a paragraph on your unknown bacteria (start the paragraph with something like “Work described in this study will identify the unknown bacterium as #####. It is….”then go on to do some background reading about your unknown and tell where it is typically found, any cool characteristics, does it cause any diseases (or have relatives that do), is it famous for anything,…etc.

Materials and Methods – Only include the media and methods for the biochemical tests you used. for the molecular test involving sequencing of the 16 S rRNA, include the colony PCR (ingredients and PCR conditions), and a brief summary of what you “did” to get a sequence (just say “the PCR was confirmed by gel electrophoresis, purified, and sequenced using the 27F primer”

Results – include figures and text. For figures, add your dichotomous key, pictures of any biochemical tests, and alignment of your sequence with the closest relative in GenBank. each figure should be numbered in the order in which they are presented in the results section text, and contain a figure legend describing the figure. The text of the results is a written narrative waking the reader through the experiments and referring to the figures, telling the reader what to look at or see.

Discussion – Now you can interpret the results, telling them what each means, basically walking the reader through the dichotomous key, and referring back to the figure that proves your point and directed you to the next test, etc. Finally interpret your molecular data (refer to the figure of the alignment) and discuss how it supports (or didn’t support) your biochemical findings. If the two didn’t match, discuss possible areas that could have led to the discrepancy.

Literature Cited – Besides your book and perhaps the Photographic Atlas for the class, try to include at least one review or scientific paper on your unknown bacteria. You don’t have to reference the protocols we gave you. Use the format given in the handout on how to write a scientific paper.