Part:BBa_K4235011:Design

Design

PROS1, the homo sapiens protein S gene, (NCBI:5627) is found on chromosome 3 and is known to exist in two major variants: the transcript variant 1 (NM_001314077.2) has an aligned length of 3,468 bp, the CDS length is 2,127 bp resulting in a 708 aa protein. The transcript variant 2 (NM_000313.4) has an aligned length of 3,372 bp, the CDS length is 2,031 bp resulting in a 676 aa protein. This part is designed from the 2,031 bp coding DNA sequence of the homo sapiens PROS1 gene, purposefully leaving out the 14 introns to streamline transcription and avoid undesired alternative splicing transcripts. The vertebrate Kozak sequence was also added to the 5’ end of the gene before synthesis.

LIC: For transforming both E coli strains we decided to use the expression vector pET His6 (2Bc-T) [1] , which contains ampicillin resistance, an IPTG inducible T7 promoter and a C-terminal 6x His tag downstream of the MCS. For cloning our insert into this vector, we used a similar LIC protocol to generate complementary overhangs on the insert and vector for the annealing reaction.
The vector has a LIC site which is acted upon by the restriction enzyme Hpa1 to linearize the vector. LIC exploits the dual polymerase - exonuclease activity of the T4 polymerase. For this LIC reaction, the insert is treated with just dGTPs and T4 polymerase, which chews back the 3’ ends until it reaches a C. Upon reaching a 3’ C, T4 polymerase gets stalled and switches its action to polymerase as it starts constantly adding dGTP. Similarly, the vector is treated with just dCTPs and T4 polymerase. This treatment step produces DNA constructs with 5’ overhangs that can anneal to one another in the final annealing reaction.