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

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

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 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

Prodigiosin Pathway G.O.I.

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

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.

References http://onlinelibrary.wiley.com/store/10.1111/j.1365- 2958.2005.04602.x/asset/j.1365- 2958.2005.04602.x.pdf?v=1&t=h6od3vl2&s=343f5137e faeab279766f7fc3f231084b43330f5 www.serratiamarcescens.net