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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.

Prepare FimE for protein expression

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