Team:LMU-Munich/Spore Coat Proteins/cloning

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[[File:SporeCoat.png|100px|right|link=Team:LMU-Munich/Spore_Coat_Proteins]]
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===Cloning Strategy===
===Cloning Strategy===
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<p align="justify">First, we used [http://partsregistry.org/Part:BBa_K823039 ''gfp''] as a proof of principle and fused it to ''cgeA'' and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823031 ''cotZ'']. This way, we can determine if it is possible to display proteins on the spore crust and if their expression has any effect on spore formation.</p>  
<p align="justify">First, we used [http://partsregistry.org/Part:BBa_K823039 ''gfp''] as a proof of principle and fused it to ''cgeA'' and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823031 ''cotZ'']. This way, we can determine if it is possible to display proteins on the spore crust and if their expression has any effect on spore formation.</p>  
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<p align="justify">Moreover, we wondered if clean deletions of the native spore crust genes would show any difference in fusion protein expression in our '''Sporo'''beads. Thus, we deleted the native ''cotZ'' and ''cgeA'' using the pMAD based gene deletion strategy described by [http://www.ncbi.nlm.nih.gov/pubmedterm=New%20Vector%20for%20Efficient%20Allelic%20Replacement%20in%20Naturally%20Nontransformable%2C%20Low-GC-Content%2C%20Gram-Positive%20Bacteria Arnaud ''et al''., 2004].</p>
<p align="justify">Moreover, we wondered if clean deletions of the native spore crust genes would show any difference in fusion protein expression in our '''Sporo'''beads. Thus, we deleted the native ''cotZ'' and ''cgeA'' using the pMAD based gene deletion strategy described by [http://www.ncbi.nlm.nih.gov/pubmedterm=New%20Vector%20for%20Efficient%20Allelic%20Replacement%20in%20Naturally%20Nontransformable%2C%20Low-GC-Content%2C%20Gram-Positive%20Bacteria Arnaud ''et al''., 2004].</p>
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<p align="justify">In the end, we were able to finish five constructs and integrated them into wild type W168 and the Δ''cotZ'' mutant:
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{| class="colored" width="100%" align="center" style="text-align:center;"
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|-
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|style="width:52%;"|
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|-
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!
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!recipient strain W168
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!recipient strain B 49 (W168 Δ''cotZ'')
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|-
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!pSB<sub>''Bs''</sub>1C-P<sub>''cotYZ''</sub>-''cotZ''-2aa-''gfp''-terminator
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|B 53
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|B 70
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|-
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!pSB<sub>''Bs''</sub>1C-P<sub>''cotYZ''</sub>-''cotZ''-''gfp''-terminator
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|B 54
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|B 71
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|-
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!pSB<sub>''Bs''</sub>1C-P<sub>''cotV''</sub>-''cotZ''-2aa-terminator
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|B 55
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|B 72
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|-
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! pSB<sub>''Bs''</sub>1C-P<sub>''cotV''</sub>-''cotZ''-terminator
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|B 56
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|B 73
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|-
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!pSB<sub>''Bs''</sub>1C-P<sub>''cgeA''</sub>-''cotZ''-2aa-terminator
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|B 52
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|B 69
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|-
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Latest revision as of 13:17, 26 October 2012

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