Part:BBa_K4604020:Design
piG_07 (tetR_mut_bluB)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 710
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1636
Design Notes
A site directed mutagenesis was done to result in a stop codon. No Methyonine triplet close to the inserted stop codon, translation should certainly end abruptly.
Cloning of piG_07
Plasmid piG_01b (BBa_K4604015) was used as a template. For the PCR we used the general protocol for the Q5 polymerase with an annealing temperature of 63°C and an elongation time of 2.5 minutes. A Dpn1 digest was done at 37°C for 2 h, afterwards the DNA was loaded onto a 1% agarose gel. The correct bands were cut out and extracted. The linear fragment was directly used for transformation, which was done according to the general protocol, the resulting colonies after an approximate 12-14h incubation time were screened by colony PCR. DNA of potential colonies containing the insert was isolated from overnight cultures (5mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing for correct insertion and no mutation.
Source
Modified piG_01b (BBa_K4604015) using Gibson Assembly.