Team:HZAU-China/Experiments

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Interlab Experiment Material and methods Plasmid construction The fragment was PCR amplified with pfu(company) or PrimeSTAR(Takara) according to product length. Product is recycled with gel extraction kit from qiagene after electrophoresis. Golden gate is conducted with total 10 liter containing 100 ng template, 5 liter buffer, 5U BsaI, and 10 U T4 ligase. With 50 cycles with 37 5min and 16 10min finished with 55 5min and deactivate at 98 for 5 min. Then transformed into trans5a using commercial component cell (xxx). homologous recombination to conducted using vyzyme xxxxx kit and operate following the guidebook. The OD measurement Cell are cultured overnight in LB broth containing corresponding antibiotics, and diluted into 1% fresh LB broth. When OD is reaching 0.6 add aTc of final concentration 200 ng/ ml to induce expression of dCas9 and inject it into 24 cell culture plate. Culture the plate in 37 and 150 rpm. Every hour put it into a plate reader to measure its OD. SDS-PAGE is conducted under the following protocol. The qPCR experiment is conducted using qPCR kit from takara. The sample is collected every 30min and freeze at liquid nitrogen after centrifugal. The genome is for qPCR is extracted using xxxx kit(takara) . Primer used for qPCR is as following. The flow cytometric is conducted with xxx. A and B are added into culture when the OD is at 0.6. After 2 hours, the riphamaycine and ceph are added to inhibit replication initiation and cell division, and cultured for another 2 hours to let the replication complete. Then the culture is stained with DAPI(roche) for 1 min and send into machine to detect the DNA concentration per cell. The Agarose Pad Assay To acquire the growth rate of bacteria for establishing the growth function, an agarose pad assay is conducted. Mix the 0.15g agarose and 10ml LB to reach the concentration of 1.5%,then heat it and add needed matter after cooling slightly.The melted agarose is supposed to be pipetted onto a cover glass(The volume is about 2.5ml.).Then put another clean cover glass on it to get a smooth flat surface after about 3 minutes. The next step is to pipette about 2.5 μL E.coli solution of 0.02 OD and get a new cover glass on it till the solution dries.It’s worth noting that maintain the favorable environment is vital.We used parafilm to avoid the evaporation and tinfoil to keep out the light when observed dCas9 4-1 which was induced by aTc. Observe the agarose pad under microscope to find the optimum bacteria, for instance, which are under the fission.The final thing you do to take and save picture 10 minutes per in 8 to 10 hours.