Team:DeNovocastrians/Experiments
Construction of biosensor G-Block
To construct absolute construct shown in figure 1 and gene less construct shown in figure 2. Genes GFP, mCherry, CatM and BenM coding sequences were ordered from twist. The coding regions CatM binding region, CatM promoter, BenM promoter/binding region and gene less counterparts were amplified out of ADP1 genome to contain overlap regions able to join catM binding to BenM binding.
Merck Hot StarTaq DNA polymerase kit with ADP1 genome and 10um of primers for each region were amplified with annealing temperature at 53°C. PCR clean up was done via QIAGEN PCR clean up DNaeasy, ultraclean, microbial kit.
Overlap extension PCR was done following optimization protocol (Hilgarth and Lanigan, 2020) with annealing temperature of 53°C for overlap stage and 57°C for amplification. Gel extraction was done via QIAGEN, QIAquick, in thyazoile orange gel, viewed under UV light. Each fragment was joined/constructed individually, to make constructs in figure 1 and 2, ie mCherry joined to catM binding region, then construct joined to CatM promoter, etc.
Restriction and ligation utilised HindIII and EcorI sites placed from primers to restrict and ligate into plasmid vector pUC19 , with 100um of vector and 300um of insert.
Transform by using thawed DH5α and 10-20ul of ligation reaction, incubate for 10 minutes on ice, heat pulse for 2 minutes, add 150ul of LB media, incubate at 37°C for 1 hour, plate on selective media.
Transform by using thawed DH5α and 10-20ul of ligation reaction, incubate for 10 minutes on ice, heat pulse for 2 minutes, add 150ul of LB media, incubate at 37°C for 1 hour, plate on selective media.
DNA Extraction
DNA of E. fergusonii was synthesized in a laboratory. DNA of Rhodococcus strain 9 and Acinetobacter baylyi ADP1 was extracted using the DNeasy ultraclean microbial kit (suggested protocol followed).
Gene Amplification
Rhodococcus strain 9 and Acinetobacter baylyi ADP1
PCR Mix: 5ul - 10x buffer for KOD + 3ul - MgSO4 (25 mM) + 5ul - dNTPs (2 mM each) + 31ul - mQ water + 1.5ul - 5' primer (10 mM) + 1.5ul - 3' primer (10mM) + 2ul - gDNA (40 ng).
Using Rhodococcus strain 9 and Acinetobacter baylyi ADP1 gDNA
Primers=
Rhodococcus strain 9:
FWD: GTACGAATTCCatgaacccatccaccac
RVS: CTAGAAGCTTtcacgacacctggacg
Acinetobacter baylyi ADP1:
FWD: AGTCGAATTCCatgacaaccctgttaaaaac
RVS: CGTCGGATCCttagctggctttgggtt
PCR run:
95ºC - 2 min
95ºC - 20 sec
54ºC - 10 sec
70ºC - 30 sec
Repeat steps 2-4 for 30 cycles
70ºC - 5 mins
hold at 12ºC
Digests
Digests were performed on benE from E. Fergusonii, Rhodococcus strain 9, Rhodococcus strain 9-his, Acinetobacter baylyi ADP1 and Acinetobacter baylyi ADP1-his.
Water bath samples at 37°C for 1 hour. Added 5ul rSAP to plasmid to prevent re-ligation. PCR cleanup was performed on samples using the QIAquick PCR and Gel Cleanup Kit.
Ligation
Ligation reactions:
Incubate at 16°C overnight.
Transformations
50ul CaCl2 competent cells + 5ul plasmid DNA (pTTQ18 control, pTTQ18::Acinetobacter baylyi ADP1 benE, pTTQ18::Acinetobacter baylyi ADP1-his benE, pTTQ18::Rhodococcus strain 9 benE, pTTQ18::Rhodococcus strain 9-his benE and pTTQ18::E. Fergusonii benE)
Incubate on ice 15 mins.
Heat shock at 42°C for 45 seconds.
Add 1ml LB to each tube.
Shake in 37°C room for 5 mins.
Spin at 1700g for 1 min.
Pour off most supernatant (leave around 100ul).
Resuspend in the remaining supernatant
Plate onto agar plates containing selective media (100ul/ml Amp)
Overnight cultures
Overnight cultures were set up for each of the transformation samples in 100ug/ml Amp M9 media. Samples were left overnight on shaking platform in 37°C room.
Plasmid Extraction
Plasmids were extracted from the overnight cultures of the transformations using the QIAprep Spin Miniprep Kit (suggested protocol was followed).
