Team:Hong Kong-CUHK/Notebook
Notebook
done by wet lab team member Heman, Savio, Billy, Greenie and Joanna
May 7 to May 15 (literature review):
1. Reviewing on sequence data of Xcm RpfC and RpfG
2. Designing primer and restriction site for the afterwards cloning
3. Designing genes fragments with restriction sites on both ends
May 16 to May 29 (literature review):
1. Codon optimization for genes on online database
June 3 to June 14 (stage 1 cloning):
1. Competent cell preparation
June 17 (stage 1 cloning):
1. PCR of synthesised gene fragments(RpfC, RpfG, Clp-TAD, Sigma 54, CBP, eforRed)
2. pRSFDuet-1 extraction from E. coli clone
June 18 (stage 1 cloning):
1. RE digestion(EcoRI, PstI) of insert (RpfC, RpfG, Clp-TAD, Sigma 54, CBP, eforRed) and vectors (pRSFDuet-1)
2. Ligation of genes to vector
3. Transformation of bacterial cloning strain, DH-5α
June 19 to 20 (stage 1 cloning):
1. Picking clones from transfromed bacteria
2. Culturing overnight
3. Collecting plasmid with clones from overnight culture
4. RE mapping to confirm extistence of cloned genes
June 21 to July 3 (stage 1 cloning):
1. Repeating procedures, starting from PCR, with the newly prepared competent cell, new sets of primers and optimised annealing temperature as no positive clone could be obtain after transformation. It was suspected that problem occurs mainly in the design of primers.
2. Collecting plasmid with gene inserts, RpfC, RpfG, Clp-TAD, Sigma 54, CBP, eforRed (stage 1 clones)
July 8 to July 12 (stage 2 and 3 cloning):
1. RE diegestion of inserts (RpfC, RpfG, Clp-TAD, Sigma 54, CBP, eforRed) by XbaI and SpeI
2. Ligation of RpfC-RpfG (A), Clp-Sigma 54 (B) and CBP-eforRed (C)
3. Ligation of A, B and C into 3 separate vectors of pRSFDuet
4. Transformation of bacterial cloning strain, DH-5α, and expression strain, BL-21
5. Picking clones from transfromed bacteria
6. Culturing overnight
7. Collecting plasmid with clones from overnight culture (stage 2 clones)
8. RE mapping to confirm extistence of cloned genes
July 15 to July 26 (stage 2 and 3 cloning):
1. PCR of construct A, B and C.
2. Adding the EcoRI, PstI restriction sites to construct A and the NdeI, XhoI restriction sites to contruct B by the primer pairs
3. RE diegestion of construct A, B and C, and the empty vector pRSFDuet-1
4. Ligation of A, B and C into 3 separate vectors of pRSFDuet, according to their respective new RE sites
5. Transformation of bacterial cloning strain, DH-5α, and expression strain, BL-21
6. Picking clones from transfromed bacteria
7. Culturing overnight
8. Collecting plasmid with clones from overnight culture (stage 3 clones)
9. RE mapping to confirm extistence of cloned genes
July 29 to August 9 (stage 4 & 5 cloning):
1. RE diegestion of construct A and the vector with construct C
2. Ligation of A into vector with construct C
3. Transformation of bacterial cloning strain, DH-5α, and expression strain, BL-21
4. Picking clones from transfromed bacteria
5. Culturing overnight
6. Collecting plasmid with clones from overnight culture (stage 4 clones)
7. RE mapping to confirm extistence of cloned genes
July 12 to August 16 (stage 4 & 5 cloning):
1. RE diegestion of construct B and the vector with construct A and C
2. Ligation of B into vector with construct A and C
3. Transformation of bacterial cloning strain, DH-5α, and expression strain, BL-21
4. Picking clones from transfromed bacteria
5. Culturing overnight
6. Collecting plasmid with clones from overnight culture (stage 5 clones)
7. RE mapping to confirm extistence of cloned genes
August 28 to August 30 (stage 4 & 5 cloning):
1. Transform final construct into BL21 competent cell
2. Starter culture and IPTG induction (1mM)
3. Collect time points 12hr, 16hr, 20hr, 24hr, 28hr
September 1 to October 4 (assays):
1. protein expression of stage 1 and 5 clones
- western blots of construct A, B and C separately
2. optimization confirmation
- western blot of RpfG protein, comparing the previous iGEM team biobrick and the RpfG biobrick optimized by our team
3. eforRed expression and signal generation
- qPCR of eforRed transcripts under 1 mM IPTG induction from the time interval of 0 to 18 hour, detecting the time-dependent transcription level
- optical density of eforRed-expressing stage 5 clones after 0-10 mM IPTG treatment, ranging from 1 to 8 hours induction time
4. DSF treatment assay
- qPCR of eforRed transcripts under 0 to 1 mM DSF treatment for 16 hour, detecting the dosage-dependent response of engineered E. Coli to DSF stimulation
5. cyclic-di-GMP suppressive drug assay
- qPCR of eforRed transcripts under 25 to 800 ug/mL azathioprine and sulfathiazole separate treatment for 16 hour, confirming the expression activation due to cyclic-di-GMP suppression