840:153g:Projects/project22

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<!-- sibboleth --><div id="lncal1" style="border:0px;"><div style="display:none;" id="id">lncal1</div><div style="display:none;" id="dtext">09/13/2012,09/20/2012,09/27/2012,10/04/2012,10/11/2012,10/18/2012,10/25/2012,11/01/2012,11/08/2012,11/15/2012,11/29/2012</div><div style="display:none;" id="page">840:153g:Projects/project22</div><div style="display:none;" id="fmt">yyyy/MM/dd</div><div style="display:none;" id="css">OWWNB</div><div style="display:none;" id="month"></div><div style="display:none;" id="year"></div><div style="display:none;" id="readonly">Y</div></div>

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Team Members

  • Stephanie Vondrak
  • Brian Hovey

Prodigiosin Production by E. Coli

Prodigiosin is a secondary metabolite of Gram-negative, gamma proteobacteria, most notably Serratia marcescens. Prodigiosin is responsible for the red color displayed by this species of bacteria. Recently, it has been discovered that prodigiosin may possess antibacterial, antifungal, antiprotozoal, antimalarial, immunosuppressive and anticancer properties. [1]

A number of genes are responsible for the production of prodigiosin (pigA-pigN), however we will only be attempting to clone the gene responsible for the start of MBC (4-methoxy-2,2-bipyrrole-5-carbaldehyde, a precursor to prodigiosin), pigI. If we are successful with this we will attempt to replicate the other genes responsible for the development of MBC. We will test for the successful uptake of pigI by SDS-PAGE.

pigI was located at NCBI and is part of a pig gene cluster accession number AJ833002.1. pigI is comprised of 1473 base pairs and since it is of bacterial origin, contains no introns. Serratia marcescens will be grown in LB and the DNA will be extracted using the protocol described in the following link: http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=2051 It will then be purified for further usage.

The pigI gene will then be amplified through PCR using designed "flanking" Biobrick compatible primers. This step will be checked by simple gel electrophoresis to ensure the target DNA was successfully amplified. pigI contains two PstI sequences which will be corrected by site-directed mutagenesis. After this, the vector of choice (psB2k3, which already has Part:BBa_I0500 - Inducible pBad/araC promoter attached) and modified pigI gene will be digested by restriction enzymes and the modified pigI gene will be inserted and ligated.

The modified vector will then be transformed into E. Coli. Success in the uptake of the vector will be checked by growing E. Coli on TSA plates with kanomycin. If this is successful, L-arabinose will be introduced to the plate to activate the pBad/araC promoter. As the product produced by pigI has no direct way of detection, a successful expression will be shown in one of two ways: 1.) The protein is successfully detected by SDS-PAGE (53.494 kDa) and/or 2.) The death of the bacteria. PigI alters proline, so tampering with this may cause lysis in the bacteria. This however, is not necessarily a positive result as the lysis could be a result of something else.


Other information:

S. marcescens prodigiosin biosynthesis gene cluster accession number - AJ833002.1 [2]

UniProt protein accession number - Q5W246 [3]

Primers modified to be Biobrick compatible:

                          Forward - 5'- GAA TTC TCT AGA ATG GCA ACC TTC ATT TCA CC - 3' 
                          Reverse - 5'- GAC GTC TGA TCA TCA TCG CGC ATT CAC CTC GG - 3'

Biobrick vector - [4]

Biobrick promoter - [5] - This is a pBad/araC promoter that prevents activation of the attached gene until L-arabinose is introduced.

Powerpoint presentation - [6]

Final Powerpoint presentation - [7]

References: Williamson, Neil R, Henrik T. Simonsen, Raef A. A. Ahmed, Gabrielle Goldet, Holly Slater, Louise Woodley, Finian J. Leeper, and George P. C. Salmond. "Biosynthesis of the Red Antibiotic, Prodigiosin, in Serratia: Identification of a Novel 2-Methyl-3-N-Amyl-Pyrrole (map) Assembly Pathway, Definition of the Terminal Condensing Enzyme, and Implications for Undecylprodigiosin Biosynthesis in Streptomyces." Molecular Microbiology. 56.4 (2005): 971-989. Print. [8]

"Serratia Marcescens." Serratia Marcescens. N.p., n.d. Web. 11 Sept. 2012. [9]

Williamson, Neil R, Henrik T. Simonsen, Raef A. A. Ahmed, Gabrielle Goldet, Holly Slater, Louise Woodley, Finian J. Leeper, and George P. C. Salmond. "Biosynthesis of the Red Antibiotic, Prodigiosin, in Serratia: Identification of a Novel 2-Methyl-3-N-Amyl-Pyrrole (map) Assembly Pathway, Definition of the Terminal Condensing Enzyme, and Implications for Undecylprodigiosin Biosynthesis in Streptomyces." Molecular Microbiology. 56.4 (2005): 971-989. Print. [10]

"Serratia Marcescens." - MicrobeWiki. N.p., n.d. Web. 11 Sept. 2012. [11]

Williamson, NR, PC Fineran, T Gristwood, SR Chawrai, FJ Leeper, and GP Salmond. "Anticancer and Immunosuppressive Properties of Bacterial Prodiginines." Future Microbiology. 2.6 (2007): 605-18. Print. [12]

Important Results and Milestones

  • 11.09.12 - 2 250ml flasks and 2 test tubes were inoculated with S. marcescens (50ml of LB media per flask and 5ml of LB media per test tube). If the flasks do not show proper growth, we will inoculate the with the contents of the test tubes.
  • 25.09.12 - DNA (or what is hoped to be DNA) was isolated and ran through a gel. Once the gel has finished, a photo will be taken.
  • 02.10.12 - Simple gel electrophoresis of our PCR did not show the expected bands. DNA samples were concentrated ten times via vacuum and will be checked via electrophoresis for increased DNA concentration.
  • 25.10.12 - We have repeatedly not gotten expected results from multiple attempts of PCR, so we are preparing for a second DNA extraction using a method from OpenWetWare.
  • 6.11.12 - We successfully extracted what appears to be DNA from S. marcescens. We ran the product through a gel and observed bright bands, so we will next digest the sample and run PCR
Recent changes

25 September 2017

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