Prodigiosin Production in E.

ColiBrian 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.

• Produced under the control of 14 genes(pigA-pigN)

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

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)

pigI GeneWe 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

Prodigiosin Pathway

G.O.I.

Gene Info

• We located the gene sequence in NCBI, with the accession number: AJ833002, and has 1473 base pairs.

• Since from bacteria, no introns

Gene Info• Extraction

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’

Removal of Internal Restriction Sites

• There are two PstI restriction sites within the gene

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

Vector and Regulator

• Vector of choice will be psB2k3

Vector and Regulator

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

Interface Vector/Gene

• Cut into vector at SpeI restriction enzyme site on plasmid

• Cut at XbaI restriction enzyme on biobrick

• Ligate

Confirmation

• The gene will be tested for by SDS-PAGE

• pigI is 53.494 kDa

Referenceshttp://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