iGEM 2016 — Team UCAS

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# Team:UCAS/Notebook

Notebook

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* [HOME](https://2016.igem.org/Team%3AUCAS)
* TEAM
  + [Team](https://2016.igem.org/Team%3AUCAS/Team)
  + [Collaborations](https://2016.igem.org/Team%3AUCAS/Collaborations)
* PROJECT
  + [Description](https://2016.igem.org/Team%3AUCAS/Description)
  + [Design](https://2016.igem.org/Team%3AUCAS/Design)
  + [Protocols](https://2016.igem.org/Team%3AUCAS/Experiments)
  + [Proof of Concept](https://2016.igem.org/Team%3AUCAS/Proof)
  + [Demonstrate](https://2016.igem.org/Team%3AUCAS/Demonstrate)
  + [Achievements](https://2016.igem.org/Team%3AUCAS/Results)
  + [Hardware](https://2016.igem.org/Team%3AUCAS/Hardware)
  + [Notebook](https://2016.igem.org/Team%3AUCAS/Notebook)
* Parts
  + [Parts](https://2016.igem.org/Team%3AUCAS/Parts)
  + [Basic Parts](https://2016.igem.org/Team%3AUCAS/Basic_Parts)
  + [Composite Parts](https://2016.igem.org/Team%3AUCAS/Composite_parts)
  + [Part Collection](https://2016.igem.org/Team%3AUCAS/Part_Collection)
* [Safety](https://2016.igem.org/Team%3AUCAS/Safety)
* [Model](https://2016.igem.org/Team%3AUCAS/Model)
* HUMAN PRACTICES
  + [Human Practices](https://2016.igem.org/Team%3AUCAS/Human_Practices)
  + [Silver](https://2016.igem.org/Team%3AUCAS/HP/Silver)
  + [Gold](https://2016.igem.org/Team%3AUCAS/HP/Gold)
* [Attributions](https://2016.igem.org/Team%3AUCAS/Attributions)

# 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 FtmOx1The 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

* [Screening of Degrading Enzymes](#js-data-notebook1)
* [Characterization of Degradation Effect and Efficiency of TetX](#js-data-notebook2)
* [Characterization of Degradation Effect and Efficiency of TetX-GFP Fusion Protein](#js-data-notebook3)
* [TA Module Selection and Kill-Switch Construction](#js-data-notebook4)
* [Circuit Construction Group](#js-data-notebook5)

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