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Characterization of Bacillus subtilis plasmid backbone created by iGEM LMU Munich 2012

The iGEM team LMU Munich 2012 has developed a plasmid backbone that is suitable for gene expression in B. subtilis (BBa_K823023). This plasmid backbone contains an integration locus in amyE, which means after being transformed into B. subtilis, the cloned insert will be integrated with B. subtilis genome at the amyE locus. B. subtilis is known for its starch degrading ability and amyE is a gene responsible for the starch degradation phenotype in B.subtilis by regulating a-amylase production.

We characterized this plasmid backbone by doing a simple growth experiment on a starch agar plate. B. subtilis strains (168 wildtype, pSac-Cm, and K823023, clockwise respectively) were inoculated in LB agar+0.4% starch and incubated overnight in 37^oC (left picture). After the addition of 0.1% iodine, the clear zone was formed around B. subtilis wildtype and B. subtilis strain containing pSac-Cm. There was no clear zone observed around B. subtilis strain containing backbone K823023 (right picture).

The inability of the B. subtilis strain containing BBa_K823023 to degrade starch has demonstrated that the amyE locus in the B. subtilis genome had been replaced. Apart from this characterization experiment, our team has been using BBa_K823023 as a plasmid backbone for our volatile detection device in B. subtilis sboA-lycopene.

References

1. Nicholson, W.L, Park, Y.K, Henkin, T.M, Won, M., Weickert, M.J, Gaskell, J.A, Chambliss, G.H. 2006. Catabolite repression-resistant mutations of the Bacillus subtilis alpha-amylase promoter affect transcription levels and are in an operator-like sequence. J Mol Biol. 1987 Dec 20;198(4):609-18.
2. Zeigler, Daniel R. 2002. Bacillus Genetic Stock Center Catalog of Strains, Seventh Edition, Volume 4: Integration Vectors for Gram-Positive Organisms. The Bacillus Genetic Stock Center. Department of Biochemistry, The Ohio State University. 484 West Twelfth Avenue,Columbus, Ohio 43210.

We also filled in their survey and now very proud that we deserved the Bavarian Medal.

iGEM LMU Munich

This iGEM team is also working on B. subtilis and therefore an ideal partner for testing each other’s constructs. They sent us 6 plasmids and 4 promoters and we are testing them for characterization. Hopefully we are able to return our constructs to them soon!

iGEM Uppsala

At the beginning of August we received the chromoproteins amilCP (blue), amilGFP (yellow) from iGEM Uppsala 2011. We were able to transform their BBa_K592009 and BBa_K592010 behind our identified promoters into E.coli. For now we are waiting untill they are transformed in B.subtilis, to check if the blue and yellow color also works in this organism. For the latest results, check our Pigment page.

Dutch iGEM teams (iGEM Wageningen, Amsterdam, Delft, Eindhoven)

All of the Dutch iGEM teams collaborated to create an iGEM presence at the Discovery Festival on Friday 28 September in Amsterdam, Rotterdam and Eindhoven. We will aim to get the public in touch with Synthetic Biology in a playful way.

Besides these Dutch iGEM meetings and the Discovery Festival, we also collaborated with Wageningen and Amsterdam on a molecular level. Many times our transformation of E.coli failed, so we ran out of the backbone biobricks for B. subtilis. Luckily Wageningen and Amsterdam were so kind to provide us with some of their bricks. Thank you guys!

iGEM Calgary 2012

Emily Hicks from the winning Calgary iGEM team in 2011, and now member of iGEM Calgary 2012, has studied in Groningen during her exchange period. She was very interested in her iGEM Groningen colleagues; therefore we asked her to give a lecture about her previous iGEM experience. We were really motivated by Emily's tips concerning medal requirements, information about wiki etc. Thank you Emily!

iGEM Virginia

Prof. Papin, supervisor to the iGEM Virginia team and supervising author to the idFBA article, graciously shared his knowledge and scripts relating to idFBA.

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