Transcript 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.
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(4methoxy-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.13652958.2005.04602.x/asset/j.13652958.2005.04602.x.pdf?v=1&t=h6od3vl2&s=343f5137e
faeab279766f7fc3f231084b43330f5
www.serratiamarcescens.net