Prodigiosin Production in E. Coli

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Prodigiosin Production in E. Coli

• Brian Hovey and Stephanie Vondrak

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What is Prodigiosin?

• A secondary metabolite of various strains of
  Serratia, and other Gram negative
  gammaproteobacteria.
• It is responsible for the red pigment produced by
  Serratia marcescens.
• Produced under the control of 14 genes(pigA-pigN)

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S. marcescens

• S. marcescens is a species of Gram negative, rod
  shaped bacteria
• Known to cause many nosocomial infections
• Thrives in high moisture environments

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Significance?

• Recently, has gotten attention for its newfound
  benefits.
• Such as antibacterial, antifungal,
  antiprotozoal, antimalarial, immunosuppressive,
  and anticancer properties
• Has no or little toxicity to cell lines (may
  operate as a cell cycle regulator)

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pigI Gene

• We chose pigI because it is involved in one of
  the beginning pathways of MBC(4-methoxy-2,2-bipyr
  role-5-carbaldehyd?)
• This is a precursor of prodigiosin

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Prodigiosin Pathway
G.O.I.
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How

• We located the gene sequence in NCBI, with the
  accession number AJ833002, and has 1473 base
  pairs.
• We will amplify the gene by PCR with primers
  (TBD)
• Amplification will be checked by gel
  electrophoresis
• Biobrick attachment
• Using BBa_I732085 Tet repressor generator.
• We will choose a plasmid with specific antibiotic
  resistances
• Some strains of S. marcescens are known to be
  resistant to a number of antibiotics naturally
• The restriction site will be cut by TBD
  restriction enzyme

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How, continued

• Hybrid DNA will be inserted into plasmid, then
  ligated.
• Plasmid will then be inserted into host bacteria
  (E. coli)
• Detection of pigI product will be determined by
  SDS-PAGE
• As a backup, we will use the Tetracycline
  repression Biobrick to test for pickup of the
  target gene
• If resistant to (TBD), then it is assumed that
  the plasmid was successfully picked up, those
  cultures can then be tested for resistance to
  tetracycline if the bacteria die, the Biobrick
  was successful and it is assumed that the gene
  also was.

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References

• http//onlinelibrary.wiley.com/store/10.1111/j.136
  5-2958.2005.04602.x/asset/j.1365-2958.2005.04602.x
  .pdf?v1th6od3vl2s343f5137efaeab279766f7fc3f23
  1084b43330f5
• www.serratiamarcescens.net