Jellyfish Green Fluorescent Protein Gene Purification and Analysis

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Jellyfish Green Fluorescent Protein Gene
Purification and Analysis

• The E. coli Kids
• Gabriel, Hilary, Jonathan Nimeka

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Summary

• The experiment of purifying and analyzing the GFP
  gene is a complicated and intensive procedure
  involving a combination of many different
  molecular biology techniques.

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Introduction
Some jellyfish have certain phenotypic
characteristics that make them glow. The protein
that does this is the Green Fluorescent Protein.
A gene in jellyfish genome encodes for this
trait. Certain techniques in genetic engineering
allow the isolation, purification and analysis of
the gene responsible for this phenomenon. The
genetic techniques we used were DNA library
transformation of E. coli, Polymerase Chain
Reaction, Restriction Enzyme Digestion, and Gel
Electrophoresis.
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Methods

• ISOLATION Plasmids from a jellyfish DNA library
  were inserted into E. coli by heat shock. The
  bacteria was cultured on petri dishes with agar
  to create colonies of glowing and non-glowing
  bacteria. A sample of each colony was then grown
  in a test tube to have enough of the DNA to work
  with.
• PURIFICATION The E. coli cells were broken open
  using a detergent and the process of boiling on a
  heat block. The cell components were extracted
  using a repetitive process of centrifuging.
  During this process the supernatant was removed
  to eventually leave only DNA molecules in the
  Eppendorf tubes.
• ANALYSIS (PCR) During PCR a mastermix was
  created to give the DNA all the components
  necessary to replicate, just as it would in a
  living cell. In other words PCR is in vitro
  replication where replication occurs in test
  tubes. PCR, however, only replicates the gene of
  interest using primers that bind to a specific
  site on the DNA template. In this case, the gene
  of interest was the gene that codes for GFP.
  

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Methods (cont..)

• ANALYSIS (RED) During restriction enzyme digest,
  restriction enzymes were added to cut the DNA
  from the gene of interest out of the plasmids in
  which they were primarily located. The enzymes
  used were PstI and HinDIII. These enzymes cut
  the plasmid into either one or two pieces.
• ANALYSIS (Gel Electrophoresis) The pure DNA
  fragments were analyzed by separating them with
  gel electrophoresis. In this process, the DNA
  molecules move along a gel due to the current
  flowing through the gel and the buffer solution.
  The DNA moves because of the negatively charged
  phosphate groups within the DNA molecules. The
  smaller fragments move faster than the larger
  ones and they were analyzed by comparing them to
  a reference.

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Results
The Polymerase Chain Reaction was not a success
because there were no DNA bands on the gel.
The Restriction Enzyme Digest was a success and
definite bands were observed on the gel.
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Photographs
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Conclusion
As a whole, the purification and analysis of the
GFP gene was successful. We didnt get the
results we wanted in PCR, but we did get the
results we wanted for Restriction Digest. The
bands were able to be identified in the samples
analyzed with restriction digest, but they were
not as clear as we hoped. The reason that PCR
didnt work was probably because we didnt have
enough DNA for PCR to be successful. Another
thing that might have gone wrong with PCR was
that any of the reagents used might have gone bad
or, for other reasons, were ineffective. Another
probable cause of the unexpected results was the
time allowed for gel electrophoresis to occur.
It is possible that if we had left the current on
longer, the results could have improved.
Overall, the experiment was a success in that we
achieved its purpose To purify and analyze the
gene that codes for GFP in jellyfish.