The document summarizes a student's IT final presentation project where they analyzed an unknown bacteriophage sample to determine its cluster assignment. The student performed experiments like plaque assays, DNA extraction, protein analysis, and mass spectrometry. By comparing the results to known cluster properties, the student concluded the phage belonged to cluster A, subcluster A3. The presentation discussed how this cluster analysis method could help schools without sequencing capabilities characterize phages.

1. IT Final Presentation
Chris Thompson
April 18th, 2013
CNIT 227

2. Table of Contents
Introduction
Materials
Methods
Results and Conclusion

3. INTRODUCTION

4. Biotechnology
Biotechnology - Any technological application that uses
biological systems, living organisms or derivatives
thereof, to make or modify products or processes for
specific use.
Biotechnology is important because without
biotechnology, many of the advancements in the latter
half of the 20th century would not have been possible,
like the Green Revolution

5. IT
IT is the biotechnology course at Purdue, focused on
researching and characterizing the properties of
mycobacteriophages.
This semester our overall goal was to develop a system
for schools without a DNA sequencer to determine the
cluster assignments of phages.

6. Bacteriophages
• A virus that infects and replicates in bacteria
• One of the most common and populous
organism in existence
• Many have a mosaic genome
• Unlimited potential usage
• Mycobacteriophages infect M.smegmatis

7. Clusters
• System to organize bacteriophages
• Phages sorted by factors such as genome
length, presence of certain genes,
organization of genome, GC content, and
plaque size and characteristics
• A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S,
Singleton, and T

8. MATERIALS

9. Materials
• Unknown phage
• M.smegmatis
• Qiagen miniprep kit
• Pressure Cycler
• Mass spectrophotometer
• BBC Proteomics Pipeline

10. METHODS

11. Organization
• HHMI gave us 15 unknown phages to work with
• Each student performed assays on their own phage
to determine its cluster assignment
• HHMI knows the cluster assignment, and will tell us if
we’re correct

12. Plaque Assays
• Created serial dilutions of phages from 10-1 to 10-10
• Let the bacteriophages infect plates of M.smegmatis
• Observed plaque sizes and characteristics
• Compared observations to known trends of clusters

13. DNA Extraction
• Used a pressure cycling machine to destroy cell
membrane and organelles
• Did a standard DNA extraction of the phage genome
using buffers and centrifuges
• Used a NanoDrop machine to analyze the
concentration of nucleotides in the sample

14. Bradford Dye
• Used Bradford Dye as a qualitative measure of
whether or not we had proteins in our sample
• My solution turned blue, so I had proteins

15. Protein Indicator Paper
• Darkness of spots shows how much protein is
present
• Darker the spot, more protein there is

16. Mass Spectrometry
• Used mass spectrometry to analyze protein products
• Went through the BBC Proteomics Discovery Pipeline
• Compared results with negative control and positive
control
• Phages that showed up the most:
– Bxz2 (13)
– EpicPhail (3)
– Rockstar (4)
– Dori (15)

17. RESULTS AND
CONCLUSION

18. Final Guess
• Cluster: A
• Subcluster: A3
• Reasons:
– Plaque size and shape looks like cluster A plaques
– Most related phages were mostly related from cluster A
and subcluster A3. EpicPhail, Rockstar, Bxz2.

19. Accomplishments
• Used complex lab procedures to analyze phage
properties
• Used mass spectrometry to discover protein
products of phage
• Were able to compare cluster properties of certain
phages to our unknown and make a prediction

20. Significance
• Cluster assignment method can be used by schools
that don’t have the funds to do full genome
sequencing
• Proves validity of undergraduate research
• Learned about biotechnology, bacteriophages,
genomes, plaques, and clusters

21. Future Work and Questions
• Was the phage novel or a common phage?
• How close was my guess?
• Is there a better method to assign clusters without
sequencing the genome?

22. The End