Team:UCAS/Notebook

Notebook

Screening of Degrading Enzymes

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

Characterization of Degradation Effect and Efficiency of TetX

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

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

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

TA Module Selection and Kill-Switch Construction

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

Circuit Construction Group

  • 14th August
    ‘pTet + RBS + GFP + DT’ was assembled on pSB1A3
  • 16th August
    ‘pT7 + RBS+ GFP + DT’ was assembled on pSB1K3. We found the E.coli bearing this plasmid is green when excited
  • 27th August
    ‘pTet + T7 RNAP’ was assembled on pSB1C3
  • 29th August
    ‘tetX-GFP’ was constructed by overlapping PCR
  • 30th August
    ‘tetX-GFP’ was assembled on pSB1C3, and was sent to be sequenced for DNA mutation
  • 4th September
    We found the ‘pTet + T7 RNAP’ plasmids we extracted lost half of pTet, when cloning ‘pTet + T7 RNAP + DT’
  • 11th September
    ‘pTet + RBS + tetX-GFP + DT’ and ‘pT7 + RBS + tetX-GFP + DT’ were assembled on pSB1C3
  • 12th September
    ‘T7 RNAP + DT + pT7 + RBS + GFP + DT’ was assembled on pSB1C3
  • 16th September
    ‘T7 RNAP + DT + pT7 + RBS + tetX-GFP + DT’ was assembled on pSB1C3
  • 20th 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.
  • 22nd September
    ‘ptet + T7 RNAP’ was assembled on pSB1A3, and was sent to be sequenced for whether half of the pTet was lost
  • 24th September
    ‘Ter(BBa_B0013) + pT7 + RBS + GFP + DT’ and ‘Ter(BBa_B0013) + pT7 + RBS + tetX-GFP + DT’ were assembled on pSB1C3.
  • 26th September
    ‘pTet + RBS + GFP + DT’ and ‘pT7 + RBS + GFP + DT’ were cloned to pSB1C3
  • 29th September
    ‘ptet + T7 RNAP + Ter + pT7 + RBS + GFP + DT’ and ‘ptet + T7 RNAP + Ter + pT7 + RBS + tetX-GFP + DT’ were assembled on pSB1C3
  • 2nd 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
  • 3rd 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
  • 7th -8th October
    The fluorescence intensity and OD600 data of Simulator, Captain Simulator, Scavenger and Captain Scavenger was got