Difference between revisions of "Part:BBa K4235011:Design"
 
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===Design===
 
===Design===
  
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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.<br>
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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.<br>
  
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This gene sequence is also codon optimized for E. coli
  
 
'''LIC protocol:'''<br>
 
'''LIC protocol:'''<br>
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For transforming both E coli strains we decided to use the expression vector pET His6 (2Bc-T) [https://www.addgene.org/37236/] , 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. <br>
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For transforming both E coli strains we decided to use the expression vector pET His6 (2Bc-T) [https://www.addgene.org/37236/],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. <br>
 
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.
 
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.
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LIC forward primer: <partinfo>BBa_K4235035</partinfo><br>
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LIC reverse primer: <partinfo>BBa_K4235036</partinfo>
  
 
===Source:===
 
===Source:===

Latest revision as of 00:23, 3 October 2022

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.

This gene sequence is also codon optimized for E. coli

LIC protocol:
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.

LIC forward primer: BBa_K4235035
LIC reverse primer: BBa_K4235036

Source:

The insert sequence was obtained by synthesis from IDT

References:

  • U.S. National Library of Medicine. (n.d.). PROS1 protein S [homo sapiens (human)] - gene - NCBI. National Center for Biotechnology Information. Retrieved August 2, 2022, from https://www.ncbi.nlm.nih.gov/gene/5627
  • Pilli, V. S., Plautz, W., & Majumder, R. (2016). The Journey of Protein S from an Anticoagulant to a Signaling Molecule. JSM biochemistry and molecular biology, 3(1), 1014.