Difference between revisions of "Part:BBa K4579008:Design"
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Because CvaC15 must end in a double glycine residue in order to be recognized and cleaved by CvaB [2], we could not use the Type 3a and 3b convention for the overhangs of our signal peptide and microcin parts. To ensure that the overhangs allow the double glycine to be maintained immediately upstream of the main microcin coding sequence, we positioned the BsaI restriction site that would be added at the 3’ end of CvaC15 such that the last 4 nucleotides of the sequence (i.e., the last nucleotide in the first glycine codon and the entirety of the second glycine codon) became the suffix for the part. We then simply used the reverse complement of those nucleotides as the prefix that would be added to the 5’ end of any microcin that was used in our microcin expression plasmid so that, when the two parts were assembled, the microcin sequence essentially “completes” the signal peptide sequence. Using NEBridge GetSet, we confirmed that this overhang did not have a significant likelihood of mis-ligating with the overhangs of our other parts. | Because CvaC15 must end in a double glycine residue in order to be recognized and cleaved by CvaB [2], we could not use the Type 3a and 3b convention for the overhangs of our signal peptide and microcin parts. To ensure that the overhangs allow the double glycine to be maintained immediately upstream of the main microcin coding sequence, we positioned the BsaI restriction site that would be added at the 3’ end of CvaC15 such that the last 4 nucleotides of the sequence (i.e., the last nucleotide in the first glycine codon and the entirety of the second glycine codon) became the suffix for the part. We then simply used the reverse complement of those nucleotides as the prefix that would be added to the 5’ end of any microcin that was used in our microcin expression plasmid so that, when the two parts were assembled, the microcin sequence essentially “completes” the signal peptide sequence. Using NEBridge GetSet, we confirmed that this overhang did not have a significant likelihood of mis-ligating with the overhangs of our other parts. | ||
Revision as of 03:26, 22 September 2023
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Because CvaC15 must end in a double glycine residue in order to be recognized and cleaved by CvaB [2], we could not use the Type 3a and 3b convention for the overhangs of our signal peptide and microcin parts. To ensure that the overhangs allow the double glycine to be maintained immediately upstream of the main microcin coding sequence, we positioned the BsaI restriction site that would be added at the 3’ end of CvaC15 such that the last 4 nucleotides of the sequence (i.e., the last nucleotide in the first glycine codon and the entirety of the second glycine codon) became the suffix for the part. We then simply used the reverse complement of those nucleotides as the prefix that would be added to the 5’ end of any microcin that was used in our microcin expression plasmid so that, when the two parts were assembled, the microcin sequence essentially “completes” the signal peptide sequence. Using NEBridge GetSet, we confirmed that this overhang did not have a significant likelihood of mis-ligating with the overhangs of our other parts.