Part:BBa_K4604027:Design
piG_K12BSb (LacIprom_riboK12_T7RBS_lacI_trcProm_sfGFP)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 215
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
If we had placed the sfGFP/marker protein directly after the riboswitch, its negative regulation in the presence of AdoCbl would have resulted in a decrease in fluorescence. In this BioBrick the riboswitch is placed in front of a repressor gene (lacI) which inturn suppresses sfGFP expression. If the riboswitch is triggered by binding of AdoCbl, the repressor expression is stopped leading to a detectable fluorescent signal.
Cloning of piG_K12BSb
Plasmid piG_K12BSa (BBa_K4604026) functioned as a template for a PCR to insert a stronger RBS from the TetR promoter (modified T7 RBS) directly behind the riboswitch. Gibson primer were designed to have the RBS as their overhang/complementary region. For the PCR we used the general protocol for the Q5 polymerase with an annealing temperature of 72°C and an elongation time of 6 minutes. A Dpn1 digest was done at 37°C overnight, afterwards the DNA was loaded onto an agarose gel. The correct bands were cut out and extracted. Gibson Assembly and AQUA cloning was used according to the protocols to assemble the plasmid. A transformation was done. As the change in the plasmid is only a RBS with a length of merely 14 bp a colony PCR was not possible to perform as screening methode, because the difference in length wouldn't have been noticeable on the gel. Instead DNA of several colonies was isolated from overnight cultures (5mL LB-medium, 50 mg/mL spectinomycin) and sent for sequencing to check for correct insertion and no mutation.