Team:UCAS/Notebook

Notebook

21^st July
The transformation of plasmid vectors carrying the tetX and cpo genes
24^th July
The overexpression and purification of CPO
25^th July
The transformation of plasmid vectors carrying the MnCcP gene and the expression of FtmOx1
26^th July
The overexpression and purification of TetX
27^th July
The expression and purification of FtmOx1
The enzymatic activity assay of CPO
29^th July
The qualitative experiment of TetX：TetX inactivates tetracycline
31^st July
The expression and purification of MnCcP
4^th August
The enzymatic activity assay of FtmOx1 and MnCcP

5^th August
The enzymatic activity assay of TetX
7^th August
The molecular reaction kinetics of Tet X
8^th August
Establishment of the method to extract tetracycline from culture medium by solid Phase extraction
10^th August
Discovered that tetracycline is oxidated by Mn(Ⅲ)
11^st -12^nd August
Tetracycline degraded by E.coli in M9 culture medium and tetracycline residues quantitative detection by LC-MS
13^rd -14^th August
Tetracycline of different concentration degraded by E.Coli in M9 culture medium
22^nd August -4^th September
Construction of the standard BioBrick TetX
10^th September
Quantitative detection of tetracycline residues with different concentration by LC-MS
11^th September
Kinetics analysis according to Michaelis-Menten Equation of MnCcP

16^th September
The qualitative experiment of tetX-GFP fusion protein
4^th October
The quantitative experiment of tetX-GFP fusion protein degradation effect
13^th October
Kinetics analysis according to Michaelis-Menten Equation of TetX

21^st -23^rd July
Selected TA modules of interes
25^th July
Used RBS Calculator to simulate the expression level of chosen toxins and antitoxins in E. coli
27^th July
Cloned 133, 134, 136, 1204, 6249 toxin from corresponding genomes by PCR, and are purified for molecular cloning
28^th July
Toxin genes of 133, 134, 136, 1204, 6249 were cloned to vector RGP-Ptac by Golden Gate
29^th July
Toxin constructions were verified by colony PCR and sequencing
4^th August
Successfully constructed plasmid PTP_Ptac
12^nd August
Measured growth curve of bacteria expressing toxin 134, 1204, 6249
14^th August
Commercially synthesized genes of toxin 5693, 5694, 5695, 5980, 4222 expressing toxin 134, 1204, 6249
20^th August
Toxin genes of 5693, 5694, 5695, 5980 were cloned to vector RGP-Ptac by Golden Gate
22^nd August
Constructed vector for antitoxin parts submission
24^th August
Toxin genes of 5694, 4222 were cloned to vector RGP-Ptac by Golden Gate
7^th September
Genes all 11 toxins were cloned to vector RGP_Ptet by Golden Gate
9^th September
Antitoxin genes of 134, 136, 1204, 6249 were cloned to vector PTP_Ptac by Golden Gate
Antitoxin genes of 134, 136, 1204, 6249 were cloned to pSB1C3-derived vector by Golden Gate
19^th September
Co-transformation of toxins and antitoxins 134, 136, 1204, 6249
22^nd September
Toxin genes of 5694 without RBS was cloned to vector RGP_Ptet by Golden Gate
24^th -27^th September
Measured growth curves of bacteria expressing toxins under TetO promoter
28^th September
The effect of antitoxins to neutralize toxins was tested qualitatively
29^th September
Constructed parts for toxins submission
3^rd October
Plasmid with TetX-GFP and antitoxin under Ptet promoter was constructed by Gibson Assembly
3^rd -6^th October
Measured growth curve of bacteria expressing toxins and corresponding antitoxins

14^th August
‘pTet + RBS + GFP + DT’ was assembled on pSB1A3
16^th August
‘pT7 + RBS+ GFP + DT’ was assembled on pSB1K3. We found the E.coli bearing this plasmid is green when excited
27^th August
‘pTet + T7 RNAP’ was assembled on pSB1C3
29^th August
‘tetX-GFP’ was constructed by overlapping PCR
30^th August
‘tetX-GFP’ was assembled on pSB1C3, and was sent to be sequenced for DNA mutation
4^th September
We found the ‘pTet + T7 RNAP’ plasmids we extracted lost half of pTet, when cloning ‘pTet + T7 RNAP + DT’
11^th September
‘pTet + RBS + tetX-GFP + DT’ and ‘pT7 + RBS + tetX-GFP + DT’ were assembled on pSB1C3
12^th September
‘T7 RNAP + DT + pT7 + RBS + GFP + DT’ was assembled on pSB1C3
16^th September
‘T7 RNAP + DT + pT7 + RBS + tetX-GFP + DT’ was assembled on pSB1C3
20^th September
We found that when amplified in the E.coli, the plasmids containing ‘T7 RNAP + DT+pT7 + RBS+GFP + DT’ and ‘T7 RNAP + DT + pT7 + RBS + tetX-GFP + DT’ would lose the parts between two DTs. The plasmids extracted from the E.coli were the mixture of plasmids carrying ‘T7 RNAP + DT + pT7 + RBS + GFP + DT’ and plasmids carrying ‘T7 RNAP + DT’ or the mixture of ‘T7 RNAP + DT + pT7 + RBS + tetX-GFP + DT’ plasmids and ‘T7 RNAP + DT’ plasmids. We thought it was because the recombination between two DTs. Thus, we decided to change the terminator of T7 RNAP.
22^nd September
‘ptet + T7 RNAP’ was assembled on pSB1A3, and was sent to be sequenced for whether half of the pTet was lost
24^th September
‘Ter(BBa_B0013) + pT7 + RBS + GFP + DT’ and ‘Ter(BBa_B0013) + pT7 + RBS + tetX-GFP + DT’ were assembled on pSB1C3.
26^th September
‘pTet + RBS + GFP + DT’ and ‘pT7 + RBS + GFP + DT’ were cloned to pSB1C3
29^th September
‘ptet + T7 RNAP + Ter + pT7 + RBS + GFP + DT’ and ‘ptet + T7 RNAP + Ter + pT7 + RBS + tetX-GFP + DT’ were assembled on pSB1C3
2^nd October
‘pTet + RBS + GFP + DT’, ‘pTet + RBS + tetX-GFP + DT’, ‘ptet + T7 RNAP + Ter + pT7 + RBS + GFP + DT’ and ‘ptet + T7 RNAP + Ter + pT7 + RBS + tetX-GFP + DT’ were cotransformed with a low-copy plasmid expressing tetR
3^rd October
The fluorescence intensity data of ‘pTet + RBS + GFP + DT’, ‘pTet + RBS + tetX-GFP + DT’, ‘pT7 + RBS + GFP + DT’, ‘pT7 + RBS + tetX-GFP + DT’ was got
7^th -8^th October
The fluorescence intensity and OD600 data of Simulator, Captain Simulator, Scavenger and Captain Scavenger was got

Team:UCAS/Notebook

Screening of Degrading Enzymes

Characterization of Degradation Effect and Efficiency of TetX

Characterization of Degradation Effect and Efficiency of TetX-GFP Fusion Protein

TA Module Selection and Kill-Switch Construction

Circuit Construction Group