Notebook
March 15th- April 13th
We noticed Igem competition and get acquainted with synthesis biology.
April 14th-May 1st
Our team, NWU-CHINA, had been organized. Then we find our PI and confirmed our project. Our PI gave us a lot of advice about following experiment.
May 3rd-May 20th
We accepted foundational lab training and learned necessary experiment operation.
June 1st-June 20th
We cleared Interlab Study requirement, RFC and 3A assembly protocol. Meanwhile, we found a lot of reference paper to find theory supporting our experiment.
July 15th-July 26th
We finished our First Interlab Study
August 1st-August 31st
Using Pak1900 to construct RFP vector and transferring constructed vector into P.a to verify expression of RFP in P.a .The detail protocol is as follows:
- We got RFP sequence from registry and cloned RFP gene by PCR. Then we separated cloned RFP
- gene by agarose gel electrophoresis and purified the gene by gel slices.
- We used pAK1900 as our vector. Then we used BamH I and Sac I to digest pAK1900 and purified gene for 5 hours.
- Then we used T4 DNA Ligase to ligated our vector and RFP gene at 4 ℃ for 24 hours
- We transferred our vector into E.coli DH5&alpha competent cell to get more vector clones. Our transferring protocol is as follows:
- Mix 4ul vector and 50ul competent cells in 1.5mL centrifuge tube at 0 ℃ for 30 min.
- Hot shock for 90 seconds at 42℃ .
- Keep competent cell at 0℃ for 4-5 min, then add 950µL Soc media into the tube.
- Incubate at 37 ℃ for 2 hours shaking at 220rpm
- Centrifuge at 6000rpm for 1min. After centrifugation, 900µL supernatant is discarded, then resuspended cells, spread the culture on plate with antibiotic 100µg/µL TC , incubate at 37℃ for 16 hours.
- Select several colonies, purified them on another selective plate, incubate at 37℃ for 12 hours.
- Incubate purified cultures with 5mL LB media and 5ul 5mg/µL TC for 5 hours.
- Using Plasmid minipreparation kit to extract the vector.
September 1st-10th
We had done our second Interlab Study. Meanwhile, we used PCR to clone GntR and AlkB2 gene. And ligated them on constructed RFP vector. Then we separated cloned gene by agarose gel electrophoresis and purified the gene by gel slices.
September 11th-October 12th
- We used Hind I, Hind II, Sal I, Hind III and Xba I, Spe I and Sal I digest pAK1900, GntR, AlkB2 for 5 hours respectively.
- Then we used T4 DNA Ligase to ligated them at 4℃ for 24 hours.
- Then we cloned our vector in DH5&alpha competent cells.
- Sequence our gene to verify our vector.
October 12th-17th
We constructed our submission parts.
