Team:LMU-Munich/Spore Coat Proteins

From 2012.igem.org

(Difference between revisions)
Line 20: Line 20:
<p align="justify">The gene ''cgeA'' is located in the ''cgeABCDE'' cluster and is regulated by its own promoter P<sub>''cgeA''</sub>. The cluster ''cotVWXYZ'' contains the gene ''cotZ'' which is cotranscribed with ''cotY'' regulated by the promoter P<sub>''cotYZ''</sub>. Another promoter of this cluster P<sub>''cotV''</sub> is responsible for the transcription of the other three genes. Those three promoters were evaluated with ''lux'' reporter genes to get an impression of their time of activation and their strength (see for more details [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks '''B'''acillus '''B'''io'''B'''rick '''B'''ox]) so they could be used for expression of spore crust fusion proteins.</p>  
<p align="justify">The gene ''cgeA'' is located in the ''cgeABCDE'' cluster and is regulated by its own promoter P<sub>''cgeA''</sub>. The cluster ''cotVWXYZ'' contains the gene ''cotZ'' which is cotranscribed with ''cotY'' regulated by the promoter P<sub>''cotYZ''</sub>. Another promoter of this cluster P<sub>''cotV''</sub> is responsible for the transcription of the other three genes. Those three promoters were evaluated with ''lux'' reporter genes to get an impression of their time of activation and their strength (see for more details [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks '''B'''acillus '''B'''io'''B'''rick '''B'''ox]) so they could be used for expression of spore crust fusion proteins.</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.  
+
<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.
 +
As we are working with B. subtilis spores, we needed to clone our final constructs into an empty Bacillus vector, so that the construct gets integrated into the genome of ''B. subtilis''. We picked the two empty vectors from our '''''Bacillus''B'''io'''B'''rick'''B'''ox, pSB<sub>BS</sub>1C for the ''cotZ'' constructs and pSB<sub>BS</sub>4S ''cgeA'' constructs. 
</p>
</p>

Revision as of 19:12, 10 September 2012

iGEM Ludwig-Maximilians-Universität München Beadzillus

Team-LMU eppis.resized.jpg

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".

IGEM HQ LMU prize.jpg

[ more news ]

Sporenfreunde