Team:Shanghai United/Notebook

Notebook

Jul. 28, 2021

1. Learnt and clarified research goals and methods as a team. Learnt safety precautions necessary for entering the laboratory.
2. Learnt and practiced the use of micropipettes.
3. Performed PCR of amilGFP gene; performed gel electrophoresis of said PCR products and tested for results.
4. Researched and learnt the reaction mechanisms of PCR and the preparation protocol of LB cultivation medium.
5. Used LB cultivation medium with KANA antibiotics to cultivate three E. coli cell lines that are KANA resistant and with ArsA, ArsD, and ArsR genes inserted, respectively.

Jul. 29, 2021

1. Performed plasmid extraction of three E. coli cell lines, cultivated since Jul. 28. Said bacteria have KANA resistance and ArsA, ArsD, or ArsR depending on the cell line. Extracted plasmids are tested for and yielded desirable concentrations.
2. Cleaned up amilGFP PCR products, obtained in Jul. 28. Cleaned up PCR products are tested for and yielded desirable concentrations.
3. Using restriction enzyme BsaI, digested ArsA, ArsD, and ArsR plasmids. Reaction mix is left overnight to react.
4. Using restriction enzymes BsaI and DpnI, digested cleaned up amilGFP PCR products. Reaction mix is left overnight to react.

Jul. 30, 2021

1. Harvested restriction enzyme digestion products of ArsA, ArsD, and ArsR plasmids as well as amilGFP PCR products; digestion was conducted in Jul. 29.
2. Performed gel electrophoresis on restriction enzyme digestion products of ArsA, ArsD, ArsR, and amilGFP genes.
3. Performed agarose gel DNA extraction of gel containing restriction enzyme digestion products. Extracted ArsA, ArsD, ArsR, and amilGFP genes are tested for and yielded suboptimal concentrations.
4. Performed T4 DNA ligase ligation of Ars genes and amilGFP genes.
5. Using ligation products of Ars genes and amilGFP genes, transformed DH5-α E. coli by heat shock method.
6. Prepared LB cultivation medium agar plates with 0.1% KANA antibiotics.
7. Spread Ars/amilGFP E. coli onto KANA antibiotics agar plates.

Jul. 31, 2021

Gel Electrophoresis Results of amilGFP PCR

Gel Electrophoresis Results of Colony PCR

1. Examined growths of Ars/amilGFP E. coli, cultivated since Jul. 30. Results were suboptimal: only 4 colonies were found in a total of 6 plates.
2. Performed colony PCR and gel electrophoresis to test for Ars/amilGFP genes in observed E. coli colonies. Only one colony resulted to be ArsD/amilGFP positive, indicating failure of experimental procedure.
3. Performed amilGFP restriction enzyme digestion on cleaned up amilGFP PCR products, obtained in Jul. 29, to prepare for first retry of experimental procedure.
4. Performed PCR of amilGFP gene to prepare for second retry of experimental procedure. Performed electrophoresis to test for amilGFP PCR products.
5. Performed gel electrophoresis to test for amilGFP restriction enzyme digestion products. Performed agarose gel DNA extraction for digested amilGFP genes.
6. Cleaned up amilGFP PCR products.
7. Performed T4 DNA ligase ligation of digested amilGFP genes and digested Ars genes, obtained in Jul. 30.
8. Cultivated three cell lines of E. coli with KANA resistance and ArsA, ArsD, or ArsR genes inserted, depending on the cell line, to prepare for second retry of experimental procedure.
9. Using ligation products of Ars genes and amilGFP genes, transformed DH5-α E. coli by heat shock method.
10. Spread Ars/amilGFP E. coli onto KANA antibiotics agar plates, prepared in Jul. 30.

Aug. 1, 2021

1. Examined growths of Ars/amilGFP E. coli, cultivated since Jul. 31. Results were optimal: colonies were observed on all six plates.
2. Performed colony PCR and gel electrophoresis to test for Ars/amilGFP genes in observed E. coli colonies. All selected colonies yielded positive results of Ars genes.
3. Extracted ArsA, ArsD, and ArsR plasmids from Ars E. coli, cultivated Jul. 31. Because the first retry of experimental procedure yielded positive results, the second retry had been terminated.
4. Cultivated Ars/amilGFP positive E. coli.

Aug. 2, 2021

1. Preserved Ars/amilGFP positive E. coli for future studies.
2. Conducted function test of Ars/amilGFP positive E. coli with different concentrations of NaH2PO4 solutions.
3. Mailed Ars/amilGFP positive E. coli samples to sequencing companies to perform Sanger sequencing of Ars/amilGFP genes.
4. Started construction of team Wikipedia reports.

Aug. 3, 2021

1. Examined function test results, conducted in Aug. 2. No green fluorescence was found in all tubes.
2. Conducted second function test of Ars/amilGFP positive E. coli with higher concentrations of NaH2PO4.
3. Continued construction of team Wikipedia.
4. Created product design information and user manual sheet for dry team for reference.

Aug. 4, 2021

1. Examined second function test results, conducted in Aug. 3. No green fluorescence was found in all tubes, indicating the inability of Ars/amilGFP E. coli to detect phosphate.
2. Analyzed results of Ars/amilGFP gene sequencing results, sequenced in Aug. 2. Constructed Ars/amilGFP genes are correct.
3. Conducted third function test of Ars/amilGFP positive E. coli with different concentrations of C2H6AsNaO2.

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