2020
July
Quorum sensing consultation (target Vibrio cholera)
Salt promoter consultation (chloride induced promoter)
pH promoter consultation (induced at pH5.5)
Design of the circuit
Colonic acid assay methodology
Literature research about antimicrobial peptides(AMPs)
Purchase chemicals and gene
Plasmid design of Lactobacillus
Purification of human lactoferrin(hLF)
Augest
Antibacterial test of hLF on V.cholera and E. coli Nissle 1917
CAI-1 quorum sensing modeling
Culture and preservation of Lactobacillus
Antibacterial Test(hLF, 7475 and kanamycin on V.cholera)
Make competent cell of Nissle 1917, Lactobacillus
Plasmid extraction of pBbE6k and pTRKH3-slpGFP
Transformation of pBbE6k into Nissle 1917
Plasmid construction of RcsB – pBbE6k(digestion and ligation)
Purification of hLF and 7475
Transformation of pBbE6k into E. coli Top10
September
Transformation of RcsB – pBbE6k and control into E. coli DH5α and Top10
Colony PCR and sequencing of Top10+pBbE6k, pBbE6k-RcsB
Purification of hLF
Sequencing of RcsB – pBbE6k and control into Top10
Plasmid extraction of pBbE6k
Transformation of pBbE6k into E. coli BL21 and E. coli Acella
Antibacterial Test
October
IPTG induced RcsB expression, electrophoresis, western blot in Acella and BL21
IPTG induced expression of RFP in Nissle 1917
Transformation of pBbE6k-RcsB into Nissle 1917
Staining and observation of BL21 and Acella with RcsB expression
Design of experiment of hLF
November~December
Transformation of RcsB into Nissle 1917
the Lactoferrin test
Verify the chloramphenicol function
2021
March
Western blot of cholera toxin detection in Vibric cholera BNCC 232030
4.1~4.9
Five schools exchange meeting
Cooperation negotiation with SZU-China
Counseling from Feiru Biotech
Counseling from doctors at the First Affiliated Hospital of Xi’an Jiaotong University
4.10~6.16
Expression and verification of RcsB in Nissle using IPTG
Construction of plasmids (CAI-1 detector, lac-ara-induced(EsaR), Lactobacillus gadR)
Counseling from doctors at the Shenzhen Second People's Hospital
Deep Blue Charity Sale
Communication with Central South University (CSU)
Counseling from scientist
Spring buds lecture
China iGEM Online Meetup
6.17~7.18
Learn and practice molecular cloning methods and other methods, including PCR, Gibson Assembly, plasmid
transformation, Western Blot, HPLC etc
Communication with the National University of Singapore (NUS)
Spring buds lecture
"Cloud West" online volunteering teaching project
7.19~7.25
Preliminary purification of colanic acid in Nissle
Transformation EsaR plasmid into BL21
7.26~8.1
Further literature research about UHPLC assay
Preparation of competent cell Acella and DH5α
Transformation of CAI-I and EsaR plasmid into Acella and gadR plasmid into DH5α
Preservation of transformed bacteria Acella and DH5α
8.2~8.8
Optimize the condition for UHPLC assay (different mobile phases)
SUSTech Hospital Exchange
Gastrointestinal meeting
8.23~8.30
Set up four standard curves of monosaccharides using UHPLC assay
Preservation of transformed bacteria DH5α with PKRTH3-ermB
PCR of arabinose promoter
PCR of vector(lac-ara-induced(EsaR))
CCiC
8.13~9.5
Optimize the condition for UHPLC assay (different flow rate, time constant, reference standards)
DNA electrophoresis for fragment(arabinose promoter) and vector Gibson
Transformation of the product of Gibson
Preservation of transformed bacteria
9.6~9.12
Optimize the condition for UHPLC assay (different gradient methods)
Western blot of PhlF in BL21
Spring buds lecture
9.13~9.19
Modification to get a better separation under more detailed conditions for UHPLC assay
PCR of the colony from Gibson
9.20~9.26
Western blot of EsaR, CqsS, LuxU, LuxO in Acella
