Team:Michigan/Notebook/Synthesis

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.