By: Vanessa Cervantes, Charles Erwing, and David Miller

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• By Vanessa Cervantes, Charles Erwing, and David
  Miller

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Introduction

• What is GFP?
• Green Fluorescent Protein (extracted from DNA of
  the jellyfish Aplysia)
• Glows under UV light
• Goals
• Isolate the sequence responsible for fluorescence
• Develop sequence with the brightest fluorescence
• Use super GFP to trace proteins
• Create FPs of different colors

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Isolating the Fluorescence Sequence
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PCR Mutagenesis

• Reduce the fidelity of the Taq DNA Polymerase
• Add 0.25 mM of MnCl2
• Increase the concentration of MgCl2.
• Reduce the dNTP prone.
• Use 20 µM of each dNTP instead of 200 µM.
• This causes the Taq DNA Polymerase to make a
  mistake.

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Fluorescence-Activated Cell Sorting FACS

• Cells are put into a tube, and allowed to drip
  down a spout at the rate of one cell at a time.
• Once the cells reach the end of the spout, a UV
  light is shined on the cell.
• A laser then determines the intensity of the
  fluorescent cell.
• Highly fluorescent cells are given a charge and
  will be attracted by a strong magnetic field into
  a separate container.
• A computer counts the cells as they fall into
  the designated tube.

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Pros and Cons of F.A.C.S.

• CONS
• Cells must be in suspension
• Does not differentiate between living or dead
  cells
• Different cell types, sizes, and shapes diffract
  the light differently

• PROS
• Separates accurately
• Saves time counting/sorting
• Able to measure light intensity more
  accurately than the naked eye

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Tracing Super GFP

• Objective easily track the protein P65 through
  the neuron of an Aplysia after its stimulated
• Amplify the Super GFP and P65 through PCR
• Ligate(connect) the two cDNA strands
• Insure that the GFP/p65 is placed before the stop
  codon
• Place the combined DNA in a virus and allow it to
  inject its genetic information into a neuron .
• Allow regular RNA transcription and translation
  to occur
• Our protein (P65, that glows) should be the
  result
• By stimulating the Aplysia we should be able to
  see the P65 move up the axon

Click on picture to play or stop animation
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Creating FPs of Different Colors

• Do PCR Mutagenesis
• Insert mutated genes into bacteria.
• Select colonies that emit different colors.
• Isolate the gene.
• Do PCR Mutagenesis to create a brighter version.

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Conclusion

• The synthesis of a super-bright GFP is made
  possible by new DNA technology in genetic
  engineering. These techniques have many new
  applications that will contribute significantly
  to the study of molecular biology.