Transcript Recombinant_Project_Proposal_revised_(really)

Prodigiosin
Production in E. Coli
Brian Hovey and Stephanie Vondrak
What is Prodigiosin?
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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)
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S. marcescens
S. marcescens is a species
of Gram negative, rod
shaped bacteria
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Grows on TSA
Known to cause many
nosocomial infections
Thrives in high moisture
environments
Sample graciously donated
by Dr. Walter
Significance?
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Recently, has gotten attention for its newfound benefits.
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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.
Gene Info
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We located the gene sequence in NCBI, with the accession
number: AJ833002, and has 1473 base pairs.
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Since from bacteria, no introns
Gene Info
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Extraction
Primers
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We will amplify the gene by PCR
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Amplification will be checked by gel
electrophoresis (pigI is 53.494 kDa)
Primers used:
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Start – 5’ ATG GCA ACC TTC ATT TCA CC 3’
End – 5’ TCA TCG CGC ATT CAC CTC GG 3’
Primers
Primer
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Primer
ATG GCA ACC TTC ATT TCA CCG ATA
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
GG CTC CAC TTA CGC GCT ACT
Primers
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Primers altered for Biobrick use:
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Start – 5’ GAA TTC TCT AGA ATG GCA ACC TTC
ATT TCA CC 3’
P
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End – 5’ GAC GTC TGA TCA TCA TCG CGC ATT
CAC CTC GG 3’
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Removal of Internal
Restriction Sites
There are two PstI restriction sites within
the gene
Removal of Internal
Restriction Sites
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5’ TC GGG CCC TTT CTG GAG GTT GAG CA 3’
5’ AG CCC GGG AAA GAC GTC CAA CTC GT 3’
5’ CTC AAG CAG TTC CTG CAG CCC AGG C 3’
5’ GAG TTC GTC AAG GAG GTC GGG TCC G 3’
Red = Mutated site
Light Blue = Complementary strand
Removal of Internal
Restriction Sites
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Mutagenesis Primers:
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Template for Mut Primer 1
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5’ – AGC CCG GGA AAG ACG TCC AAC TCG T 3’
Complimentary for Mut Primer 1
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5’ – TCG GGC CCT TTC TGG AGG TTG AGC A 3’
Removal of Internal
Restriction Sites
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Template for Mut Primer 2
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5’ – CTC AAG CAG TTC CTG CAG CCC AGG C 3’
Complimentary for Mut Primer 2
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5’ – GAG TTC GTC AAG GAG GTC GGG TCC G 3’
Vector and Regulator
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Vector of choice will be psB2k3
Vector and Regulator
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Regulator will be Part:BBa_I0500 Inducible pBad/araC promoter (expose to
arabinose to activate)
Interface Vector/Gene
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Cut into vector at SpeI restriction enzyme
site on plasmid
Cut at XbaI restriction enzyme on biobrick
Ligate
Confirmation
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The gene will be tested for by SDS-PAGE
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pigI is 53.494 kDa
References
http://onlinelibrary.wiley.com/doi/10.1111/j.13652958.2005.04602.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