Part:BBa_K245050:Design
GyrB-CutA1
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 428
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 100
Illegal AgeI site found at 238 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
The part BBa_K245116 codes for the 24 kDa N-terminal GyrB fragment of gyrase subunit B, and the part BBa_K245129 codes for CutA1, a copper binding protein, which are linked by a friendly scar tccggc (SG). Coding sequence for Gyrase subunit B fragment was amplified by PCR from a plasmid. Coding sequence for CutA1 was multiplied by colony PCR from E. coli genomic DNA. Both genes were cloned into the functionalized vector (BBa_K245005) carrying T7 promoter, RBS, ATG at 5' and T7 terminator and STOP codon at 3’ of multiple-cloning site. In this version of vector a sequence coding for His-tag is inserted between ATG and multiple-cloning site, which results in His-tag at the N-terminus of the protein product of the gene. In this way two basic parts were obtained: BBa_K245116 coding for GyrB and BBa_K245129 coding for CutA1.
The basic BioBrick part coding for GyrB (BBa_K245116) was cut with BspEI and PstI restriction enzymes creating back vector and basic part coding for CutA1 (BBa_K245129) was cut with NgoMIV and PstI restriction enzymes creating back insert. Ligation of back vector and back insert resulted in generation of a composite part called GyrB-CutA1 (BBa_K245050, Figure 1), where CutA1 coding sequence is at the 3’ and GyrB coding sequence is at the 5’ of the composite part. The friendly scar between basic parts BBa_K245116 and BBa_K245129 is tccggc (SG). Due to design of our functionalized vector polypeptide product of composite part (BBa_K245050) contains a His-tag on N-terminus.
Source
Coding sequence for Gyrase subunit B fragment was amplified by PCR from a plasmid. Coding sequence for CutA1 was multiplied by colony PCR from E. coli genomic DNA.