Team:LMU-Munich/Spore Coat Proteins

From 2012.igem.org

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[[File:Imamura, 2011 & McKenney, 2010.png|Protein distribution in spore coat of ''Bacillus subtilis''|thumb|610px]]
 
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<p align="justify">The first step was to fuse ''gfp'' to ''cgeA'' and ''cotZ'' as a proof of principle. This way we would determine if it is possible to display proteins on the spore crust and if their expression has any effect on spore formation. Therefore we first fused ''cotZ'' to its two native promoters, P<sub>''cotV''</sub> and P<sub>''cotYZ''</sub>, and to P<sub>''cgeA''</sub>, which regulates the transcription of ''cgeA''. For cgeA we only used its native promoter P<sub>''cgeA''</sub> and the stonger one of the two promoters of the ''cotVWXYZ'' cluster, P<sub>''cotYZ''</sub>. While ''gfp'' was ligated to B0014, a terminator. When these constructs were finished and confirmed by sequencing, we fused them together applying the Freiburg standard to create in frame fusion proteins, flanked by one of the three promoters and the terminator.</p>  
<p align="justify">The first step was to fuse ''gfp'' to ''cgeA'' and ''cotZ'' as a proof of principle. This way we would determine if it is possible to display proteins on the spore crust and if their expression has any effect on spore formation. Therefore we first fused ''cotZ'' to its two native promoters, P<sub>''cotV''</sub> and P<sub>''cotYZ''</sub>, and to P<sub>''cgeA''</sub>, which regulates the transcription of ''cgeA''. For cgeA we only used its native promoter P<sub>''cgeA''</sub> and the stonger one of the two promoters of the ''cotVWXYZ'' cluster, P<sub>''cotYZ''</sub>. While ''gfp'' was ligated to B0014, a terminator. When these constructs were finished and confirmed by sequencing, we fused them together applying the Freiburg standard to create in frame fusion proteins, flanked by one of the three promoters and the terminator.</p>  
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[[File:Final construct-2.png|Scheme of variants of the final fusion constructs Promoter-Gene-GFP-Terminator|thumb|610px]]
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<font color="#EBFCE4">Scheme of variants of the final fusion constructs Promoter-Gene-GFP-Terminator</font>
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<p align="justify">As we are working with B. subtilis spores, we needed to clone our final constructs into an empty Bacillus vector, so that they could get integrated into the genome of ''B. subtilis'' after transformation. Thus we picked the two empty vectors from our '''''Bacillus''B'''io'''B'''rick'''B'''ox, pSB<sub>BS</sub>1C for ''cotZ'' constructs and pSB<sub>BS</sub>4S for ''cgeA'' constructs. While the integration of pSB<sub>BS</sub>1C-''cotZ'' constructs was checked via a starch test, the pSB<sub>BS</sub>4S''-cgeA'' constructs were tested with threonin-deficient agar plates. The clones with the right integrated constructs have then been chosen for further analysis.</p>
<p align="justify">As we are working with B. subtilis spores, we needed to clone our final constructs into an empty Bacillus vector, so that they could get integrated into the genome of ''B. subtilis'' after transformation. Thus we picked the two empty vectors from our '''''Bacillus''B'''io'''B'''rick'''B'''ox, pSB<sub>BS</sub>1C for ''cotZ'' constructs and pSB<sub>BS</sub>4S for ''cgeA'' constructs. While the integration of pSB<sub>BS</sub>1C-''cotZ'' constructs was checked via a starch test, the pSB<sub>BS</sub>4S''-cgeA'' constructs were tested with threonin-deficient agar plates. The clones with the right integrated constructs have then been chosen for further analysis.</p>

Revision as of 13:51, 17 September 2012

iGEM Ludwig-Maximilians-Universität München Beadzillus

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The LMU-Munich team is exuberantly happy about the great success at the World Championship Jamboree in Boston. Our project Beadzillus finished 4th and won the prize for the "Best Wiki" (with Slovenia) and "Best New Application Project".

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