Prodigiosin Production in E. Coli

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. Primers • ATG GCA ACC TTC ATT TCA CCG ATA CTG GAG GCC CTG TTC… • …AAG GTG GAC AGA GGA CGT TTG TCC GAG GTG AAT GCG CGA TGA ATG GCA ACC TTC ATT TCA CCG ATA Primer GG CTC CAC TTA CGC GCT ACT Primer

11. Primers • Primers altered for Biobrick use: • Start – 5’ GAA TTC TCT AGA ATG GCA ACC TTC ATT TCA CC3’ • End – 5’ GAC GTC TGA TCA TCA TCG CGC ATT CAC CTC GG 3’ E X P S

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

13. Removal of Internal Restriction Sites • 5’ AG CCC GGG AAA GAC GTCCAA CTC GT 3’ • 5’ CTC AAG CAG TTC CTG CAG CCC AGG C 3’ 5’ TC GGG CCC TTT CTG GAG GTT GAG CA 3’ 5’ GAG TTC GTC AAG GAG GTC GGG TCC G 3’ Red = Mutated site Light Blue = Complementary strand

14. Removal of Internal Restriction Sites • Mutagenesis Primers: • Template for Mut Primer 1 • 5’ – AGC CCG GGA AAG ACG TCC AAC TCG T 3’ • Complimentary for Mut Primer 1 • 5’ – TCG GGC CCT TTC TGG AGG TTG AGC A 3’

15. Removal of Internal Restriction Sites • Template for Mut Primer 2 • 5’ – CTC AAG CAG TTC CTG CAG CCC AGG C 3’ • Complimentary for Mut Primer 2 • 5’ – GAG TTC GTC AAG GAG GTC GGG TCC G 3’

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

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

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

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

20. 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