Molecular Biology Lab SCIENTIFIC POSTERS Sp 2009:
Using 16s rDNA sequence to identify unknown microorganisms
To be presented in class on Nov 23 & 24 and Dec 2
(our Final Exam will be Nov 30 and Dec 1 & 2)
General Guidelines:
The easiest way to do the poster is to break it up into pieces and everybody in
the group take one or two pieces. Group leaders are responsible for assigning
tasks and coordinating the work of putting the poster together. They are not
responsible for doing all the work themselves! Produce a poster that you will be
happy to have displayed in the hall.
Please remember that everybody in the group is responsible for all the
information on the poster. Be sure you all understand what was done.
Group One
(group leader)
Group Two
(group leader)
Posters are similar to journal articles, but in a condensed way. All the
information has to be easy to understand and presented in short segments on
the poster. This is the most significant difference between a poster and a journal
article. You can use headings with a few main points explained, short
paragraphs, or other ways to break up the text. In the Methods, you may want to
use an outline or a table. The font should be large and easy to read (no script!).
The poster can be colorful, in moderation. It should not be distracting or
challenging to your audience. We will prepare a “power point poster”, where the
contents of the poster are on a giant power point slide. This is the method most
commonly used now to prepare a poster. You will see many examples in the
hall, and this is a good skill to learn.
The main parts of a poster are:
1. Title:
Includes name of project, people who did the work (your group) and location.
Feel free to change the title that I used above.
2. Introduction:
Explain why the project was performed (similar to a "statement of purpose" when
writing a paper). Like a paper, you need to give background information so
people can understand the results. We had one main goal, identification of
unknown bacterial samples by using DNA sequencing. But we had a reason for
choosing a certain segment of DNA.
PCR of 16s rDNA
Posters need to explain the basics about the PCR technique and how it can be
used to amplify one small segment of a genome. In our case, it was a portion of
the bacterial 16s rDNA gene. What is the significance of that segment? How do
researchers use sequence information to identify microorganisms and to
establish phylogenetic relationships? It will be important to give enough
background so people reading the poster in the hall will understand the use of
ribosomal DNA in identification.
Sequencing
Briefly explain the process of "dideoxy sequencing". You might also want to
cover how modern capillary sequencers work. That is the type of sequencer at
Grice and at Clemson.
Identification
How did we identify our unknown bacteria? Explain the things we looked for
when examining our chromatograms, such as N bases or two bases assigned to
one peak. Describe how we determined the identity of our unknowns. What was
the purpose of using the BLAST program at NCBI? What information did we get
from the BLAST search? Why did we also use the RDB?
Remember that figures are a good way to explain things without having big
blocks of text. So you might consider incorporating figures, charts, diagrams, etc.
into the Introduction. Some figures might serve double duty in other sections
such as Methods.
As a caution, I urge you to not get all your information from the internet. Make
sure your information can be referenced to a text or journal article or other
reputable scientific source.
3. Methods:
To recap, our Methods for identifying the rDNA PCR segment included:
Collecting bacterial samples, isolation of bacterial DNA (Qiagen kit), PCR,
verification of PCR product (run gel), purification of PCR product from agarose
gel (Qiagen), verification of purification (concentration determined by
spectrophotometry), and sending samples to the sequencing facility. They did the
"cycle sequencing" reaction, which is PCR using the dideoxy nucleotides.
Samples were run on a capillary sequencer.
This is a lot of stuff! So refer to existing protocols when you can. Also, summarize
information (tables, charts, etc.). Check journal articles for ideas on how to
present Methods information. In particular, there is a certain format that is
followed when referring to a kit or reagent from a company. Check the Methods
section of a journal article and follow that format.
The experiment starts with PCR of a certain region of the 16s rDNA gene. This
is a useful link for the sequence of the “Lycoming” primers (from Dr Jeff Newman
at Lycoming College) http://srv2.lycoming.edu/~newman/rrna/primer.htm .
Another useful link from Dr Newman:
http://srv2.lycoming.edu/~newman/rrna/rrnalab.htm .
Then the DNA was handed over to the sequencing facility. A proper way to
designate this would be: Genewiz Inc. (www.genewiz.com).
Finally, we used the public database at NCBi and the Ribosomal Database
project for our identification, and the Biology Workbench (at the UCSD
Supercomputing Center) to make a phylogenetic tree.
4. Results:
A figure should have a Figure Number, probably a title, and a Figure Legend.
The Figure Legend will tell what the lanes on a gel represent, what the axes on
the graphs are, etc. Tables should have a number and a title. I suggest you look
around the building at posters to see how data is presented. All posters need a
figure or table that summarizes the identification results. It should include the
unknown code # and identification to at least the genus level.
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You can include photos of petri plates, water fountains, PCR machine, etc.
if you want. It is up to the group what to include on their poster.
Include gel photos large enough to see the important points. The gel
pictures should be properly labeled. I have a gel photo of the preparative
gel before we cut out the PCR band and I can send it to you via email.
A sequence printout (chromatogram) can be made using an inkjet printer.
Please include a phylogenetic tree for your entire section.
5. Discussion or Conclusions:
Group leaders, please consider these questions with your group. Some of
them are thinking questions and I would like to have some discussion in class.
In general, how successful was our approach for identifying unknown bacteria?
 Of the bacteria that we identified, does it make sense that they would be
found at water fountains or keyboards or the other surfaces that we
examined?
 We would like to be able to identify unknown bacteria based on sequence
information. We also discussed several journal articles that used the
traditional methods used for bacterial identification. What are the
advantages and disadvantages of each approach?
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In some cases, the sequence that we BLASTed, even after “cleaning up”,
was highly related to two different genera. This makes an accurate
identification of our unknown a difficult task. Are there ways that we might
improve our chances of obtaining the correct identification? What are they
and how would they help? (Suggest ways that we might be able to
improve this aspect of the experiment.)
What is the difference between a rooted and an unrooted tree? Could we
also improve the experiment’s phylogenetic analysis?
What are the pros and cons of using public databases?
6. References:
Use any accepted format. Reference thoroughly. If you do use a (reputable)
web source, give the web address (the URL).
This is the information I would like on everyone's poster. But please feel free to
elaborate on topics that interest you! I am looking forward to learning more from
your posters. So have some fun with this assignment, and take advantage of the
opportunity to expand your horizons.
7. Poster Presentation:
Each group will present its poster in class, and the other group will be the
audience. You will present the section(s) of the poster that you worked on.
Please practice your presentation so you will be confident in what you say.
Ideally, everyone should practice with the group so you can coordinate what
each person will say. This is important, because sometimes Methods material is
discussed in Intro, or Results are discussed in Methods. That's OK, as long as
you leave your colleagues with something to talk about! Otherwise they are left
standing with nothing to say – not good! So work on this ahead of time and
practice your part so it flows smoothly.
Your presentation will be a reflection of how well you understand your section
and the project as a whole, so show your audience that you know what's going
on!
Public speaking is an important skill for a scientist to have, but it makes many
people nervous. Practice is the best remedy. So practice by yourself and with the
group. Don't be nervous when you give your part because you will be speaking
before a very friendly and understanding audience.