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Prodigiosin Production in E. Coli

Prodigiosin Production in E. Coli. Brian Hovey and Stephanie Vondrak. 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.

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Prodigiosin Production in E. Coli

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  1. Prodigiosin Production in E. Coli • Brian Hovey and Stephanie Vondrak

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

  3. S. marcescens • S. marcescens is a species of Gram negative, rod shaped bacteria • Grows on TSA • Known to cause many nosocomial infections • Thrives in high moisture environments • Sample graciously donated by Dr. Walter

  4. 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)

  5. pigI Gene • We chose pigI because it is involved in one of the beginning pathways of MBC(4-methoxy-2,2`-bipyrrole-5-carbaldehydе) • This is a precursor of prodigiosin

  6. Prodigiosin Pathway G.O.I.

  7. Gene Info • We located the gene sequence in NCBI, with the accession number: AJ833002, and has 1473 base pairs. • Since from bacteria, no introns

  8. Gene Info • Extraction

  9. Primers • We will amplify the gene by PCR • Amplification will be checked by gel electrophoresis (pigI is 53.494 kDa) • Primers used: • Start – 5’ ATG GCA ACC TTC ATT TCA CC3’ • End – 5’ TCA TCG CGC ATT CAC CTC GG 3’

  10. Removal of Internal Restriction Sites • There are two PstI restriction sites within the gene

  11. Removal of Internal Restriction Sites • We will create primers that contain a changed nucleotide so the replicated strands do not contain the RE sites • Strands will overlap to create a new altered segment

  12. Vector and Regulator • Vector of choice will be psB2k3

  13. Vector and Regulator • Regulator will be Part:BBa_I0500 - Inducible pBad/araC promoter (expose to arabinose to activate)

  14. Interface Vector/Gene • Cut into vector at SpeI restriction enzyme site on plasmid • Cut at XbaIrestriction enzyme on biobrick • Ligate

  15. Confirmation • The gene will be tested for by SDS-PAGE • pigI is 53.494 kDa

  16. References • http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2005.04602.x/pdf • www.serratiamarcescens.net • http://mic.sgmjournals.org/content/150/11/3547.long#ref-46 • http://microbewiki.kenyon.edu/index.php/Serratia_marcescens • http://www.ncbi.nlm.nih.gov/pubmed/18041902

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