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 produced by 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 CC 3’ 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 XbaI restriction 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#re f-46 http://microbewiki.kenyon.edu/index.php/Serratia_marc escens http://www.ncbi.nlm.nih.gov/pubmed/18041902