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Team:Michigan/Notebook
From 2012.igem.org
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<p style="font-family:Courier, monospace;"><span style="color:red;">(TAA TAA)</span> T <span style="background-color:#C8E0DB;">ACTAGT</span> A GCGGCCG <span style="background-color:#A9C9A4;">CTGCAG</span></p> | <p style="font-family:Courier, monospace;"><span style="color:red;">(TAA TAA)</span> T <span style="background-color:#C8E0DB;">ACTAGT</span> A GCGGCCG <span style="background-color:#A9C9A4;">CTGCAG</span></p> | ||
| - | <p><br/>The suffix is RFC10 - we don’t need it to be RFC25 because two stop codons not in the original suffixes have been introduced before the suffix starts. LVA-mediated degradation depends on a series of amino acids at the C-terminus binding to cellular degradation machinery, and failure to abruptly halt transcription after this sequence may result in improper binding and degradation. Unlike in the RFC25 suffix, which adds linker amino acids after the protein domain ends, RFC10 adds a tyrosine before its internal stop codon, assuming translation proceeds that far-this could severely hinder binding.</p> | + | <p><br/>The suffix is RFC10 - we don’t need it to be RFC25 because two stop codons not in the original suffixes have been introduced before the suffix starts. LVA-mediated degradation depends on a series of amino acids at the C-terminus binding to cellular degradation machinery, and failure to abruptly halt transcription after this sequence may result in improper binding and degradation. Unlike in the RFC25 suffix, which adds linker amino acids after the protein domain ends, RFC10 adds a tyrosine before its internal stop codon, assuming translation proceeds that far - this could severely hinder binding.</p> |
</div> | </div> | ||
</div> | </div> | ||
| + | <p class="title">Main tasks:</p> | ||
| + | |||
| + | <ol> | ||
| + | <li>Extract Hbif and the degradation tags from their parent plasmids, place in biobrick-compatible plasmids.</li> | ||
| + | <li>Obtain RFC25-standard plasmid Bba-K157000 for use in construction.</li> | ||
| + | <li>Clone the LVA tag onto Hbif’s C-terminus.</li> | ||
| + | <li>Design primers to FimE which place an AgeI site at it’s C-terminus.</li> | ||
| + | <li>Clone the LVA tag onto the modified FimE.</li> | ||
| + | <li>Assay functionality of both. </li> | ||
| + | </ol> | ||
<h2>Prepare FimE for protein expression</h2> | <h2>Prepare FimE for protein expression</h2> | ||
Revision as of 00:25, 6 July 2012
Team:Michigan/Notebook/Protocols
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Increasing Recombinase HbiF and FimE Turnover Rate with LVA Degradation Tag
HbiF and LVA degradation tag were ordered from IDT with the following specifications.
EcoRI
XbaI
SpeI
PstI
AgeI
NgoMIV
HbiF possesses an N-terminus in the (truncated) RFC10 format :
GAATTC GCGGCCGC T TCTAG [ATG]
No extra amino acids are created by this prefix, in comparison to the standard RFC25 prefix, which creates a serine and glycine. The suffix proceeds as follows:
ACCGGT TAAT ACTAGT A GCGGCCG CTGCAG
In order to properly attach the LVA degradation tag, the prefix of the tag sequence must be complementary to the suffix of Hbif. For this part, the prefix and suffix are reversed in comparison to above: the prefix is RFC25, and the suffix is RFC10, for reasons that are discussed below.
Prefix:
GAATTC GCGGCCGC T TCTAGA TG GCCGGC
Suffix:
(TAA TAA) T ACTAGT A GCGGCCG CTGCAG
The suffix is RFC10 - we don’t need it to be RFC25 because two stop codons not in the original suffixes have been introduced before the suffix starts. LVA-mediated degradation depends on a series of amino acids at the C-terminus binding to cellular degradation machinery, and failure to abruptly halt transcription after this sequence may result in improper binding and degradation. Unlike in the RFC25 suffix, which adds linker amino acids after the protein domain ends, RFC10 adds a tyrosine before its internal stop codon, assuming translation proceeds that far - this could severely hinder binding.
Main tasks:
- Extract Hbif and the degradation tags from their parent plasmids, place in biobrick-compatible plasmids.
- Obtain RFC25-standard plasmid Bba-K157000 for use in construction.
- Clone the LVA tag onto Hbif’s C-terminus.
- Design primers to FimE which place an AgeI site at it’s C-terminus.
- Clone the LVA tag onto the modified FimE.
- Assay functionality of both.
Prepare FimE for protein expression
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