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Team:Grinnell/Project/RsaA
From 2011.igem.org
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| - | <p>RsaA is the surface-layer (S-layer) protein produced by the gram-negative bacteria <i>Caulobacter crescentus</i>. It covers the surface of the cell in a 2-D hexameric crystal and accounts for 10-12% of total protein synthesis by the cell | + | <p>RsaA is the surface-layer (S-layer) protein produced by the gram-negative bacteria <i>Caulobacter crescentus</i>. It covers the surface of the cell in a 2-D hexameric crystal and accounts for 10-12% of total protein synthesis by the cell (<a href="https://2011.igem.org/Team:Grinnell/Attributions#Awram">Awram 1998</a>). Key regions of the protein include the N-terminal region, which is necessary for binding of the protein to the surface of the cell, and the C-terminal region, which acts as a secretion tag (<a href="https://2011.igem.org/Team:Grinnell/Attributions#Bingle">Bingle 2000</a>). |
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| + | The secretion pathway for RsaA is a robust ATP-dependent type I secretion pathway. These features of RsaA and its secretion pathway allows us to take advantage of it by creating chimeric proteins with the C-terminal secretion tag. These proteins should then be expressed and secreted in <i>Caulobacter</i> in relatively high yield. Another advantage of this is that <i>Caulobacter</i> secretes very few proteins, so isolation of the desired protein from liquid culture supernatant should be relatively easy.</p> | ||
<p>There is a limit on the effectiveness of expression of this type of recombinant protein. The main limiting factors are the size of the protein joined to the C-terminal of RsaA (smaller protein regions are better expressed and secreted) and the codons used in the gene in question (<i>Caulobacter</i> has a GC rich genome). In addition to addressing these issues, use of a strong constitutive promoter can help. In <i>Caulobacter</i> using the P<sub>rsaA</sub> promoter is effective, and becomes more effective if native RsaA production is turned off.</p> | <p>There is a limit on the effectiveness of expression of this type of recombinant protein. The main limiting factors are the size of the protein joined to the C-terminal of RsaA (smaller protein regions are better expressed and secreted) and the codons used in the gene in question (<i>Caulobacter</i> has a GC rich genome). In addition to addressing these issues, use of a strong constitutive promoter can help. In <i>Caulobacter</i> using the P<sub>rsaA</sub> promoter is effective, and becomes more effective if native RsaA production is turned off.</p> | ||
Revision as of 23:51, 28 September 2011
RsaA
RsaA is the surface-layer (S-layer) protein produced by the gram-negative bacteria Caulobacter crescentus. It covers the surface of the cell in a 2-D hexameric crystal and accounts for 10-12% of total protein synthesis by the cell (Awram 1998). Key regions of the protein include the N-terminal region, which is necessary for binding of the protein to the surface of the cell, and the C-terminal region, which acts as a secretion tag (Bingle 2000).
