Part:BBa_K4604018:Design
piG_02b (tetR_riboK12_mazF_mTurq)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 710
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 883
Illegal AgeI site found at 1376 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2250
Design Notes
Site directed mutagenesis was done between the tet promoter and RBS to remove a EcoRI restriction site. The region downstream of the tetA/R promoter was shortened (in comparison to piG_01a/BBa_K4604016) to counteract the leakiness.
Cloning of piG_02b
Plasmid piG_02a (BBa_K4604017) was used as a template. For the PCR we used the general protocol for the Q5 polymerase with an annealing temperature of 56°C and an elongation time of 5 minutes. A Dpn1 digest was done at 37°C for an hour, afterwards the DNA was loaded onto a 1% agarose gel. The correct bands were cut out and extracted. Gibson Assembly was used according to the protocol to assemble the plasmid. A transformation was done and 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_02a (BBBa_K4604017) using Gibson Assembly.