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
The lacI promoter, lacI gene, trc promoter, sfGFP and rrnB terminator were given to us from iGEM Freiburg 2022 on a plasmid called pIG23_04. We took plasmid pIG23_03 (also given to us from iGEM Freiburg 2022) as our backbone, including an antibiotic resistance (spectinomycin) gene and Ori. SfGFP, trc promoter, lacI and lacI promoter were taken from pIG23_04 and cloned into pIG23_03 using Gibson Assembly and AQUA cloning. Both plasmids were templates for PCR, to amplify the parts and add the correct overhangs via gibson primers. After assembling the two plasmid parts, this new plasmid, the sensor backbone, was again used as PCR template to clone the correct overhangs for the insertion of the riboswitch between lacI promoter and lacI gene. The sequence for riboswitch K12 originates from E. coli K12 from the 5´-UTR of the btuB gene. We ordered the DNA for the K12 riboswitch from IDT and used it as a template. For the PCR we used the general protocol for the Q5 polymerase with varying parameters (elongation time and annealing temperature):
| fragment | Annealing temp. | Elongation time | Fragment size (in bp) |
|---|---|---|---|
| trc promoter, sfGFP, lacI promoter, lacI gene | 72°C | 1.5 min | 2670 |
| backbone (Ori and resistance) | 72°C | 1.5 min | 2080 |
| K12 riboswitch | 65°C | 30 s | 250 |
| create overhangs to add riboswitch between lacI prom. and lacI | 62°C | 6 min | 4700 |
The DNA of the plasmid fragments, pIG23_04 and pIG23_03, was loaded onto an agarose gel. The correct bands were cut out and extracted. Plasmid fragments of pIG23_03 and pIG23_04 were first assembled to form the sensor backbone with Gibson Assembly and AQUA cloning, according to the protocols. 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, 50 mg/mL spectinomycin ) and sent for sequencing for correct insertion and no mutation.
Then with the sensor backbone as a template the PCR was performed to add the correct overhangs to insert the riboswitch between lacI promoter and lacI. A Dpn1 digest was done at 37°C for an hour, afterwards the DNA was loaded onto an agarose gel. The correct bands were cut out and extracted. The PCR for the riboswitch was also performed, subsequently, the DNA was loaded onto an agarose gel. The correct bands were cut out and extracted. Riboswitch and sensor backbone were assembled via Gibson Assembly and AQUA cloning according to the protocols. 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, 50 mg/mL spectinomycin ) and sent for sequencing for correct insertion and no mutation.