IT Final PresentationChris ThompsonApril 18th, 2013

CNIT 227

Introduction

Materials

Methods

Results and Conclusion

Table of Contents

INTRODUCTION

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

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.

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

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

MATERIALS

Materials

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

METHODS

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

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

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

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

Protein Indicator Paper

• Darkness of spots shows how much protein is present

• Darker the spot, more protein there is

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)

RESULTS AND CONCLUSION

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.

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

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

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?

The End