iGEM 2019 — Team AHUT_China

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# Team:AHUT China/Notebook

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* [HOME](https://2019.igem.org/Team%3AAHUT_China)
* TEAM

  [Team Members](https://2019.igem.org/Team%3AAHUT_China/Team)
  [Attributions](https://2019.igem.org/Team%3AAHUT_China/Attributions)
  [Collaboration](https://2019.igem.org/Team%3AAHUT_China/Collaborations)
* PROJECT

  [Description](https://2019.igem.org/Team%3AAHUT_China/Description)
  [Design](https://2019.igem.org/Team%3AAHUT_China/Design)
  [Safety](https://2019.igem.org/Team%3AAHUT_China/Safety)
  [Demonstrate](https://2019.igem.org/Team%3AAHUT_China/Demonstrate)
  [Medals](https://2019.igem.org/Team%3AAHUT_China/Medals)
* EXPERIMENT

  [Result](https://2019.igem.org/Team%3AAHUT_China/Result)
  [Notebook](https://2019.igem.org/Team%3AAHUT_China/Notebook)
* PARTS

  [Overview](https://2019.igem.org/Team%3AAHUT_China/Parts)
  [New Parts](https://2019.igem.org/Team%3AAHUT_China/New_parts)
  [Improved Parts](https://2019.igem.org/Team%3AAHUT_China/Improved_parts)
  [Characterization](https://2019.igem.org/Team%3AAHUT_China/Characterization)
* [HUMAN PRACTICES](https://2019.igem.org/Team%3AAHUT_China/Human_Practices)
* [JUDGING FORM](https://igem.org/2019_Judging_Form?id=2949)

# Notebook

## The notebook recorded the whole process of our experiment.

READING TIME:

* # WEEK 1

  This week we measured the standard curves of two particles, compared them, sent the results of
  the second measurement datas to the measurement committee for verification, hoping to find the
  problem in time for correction.

  ![WEEK1_1](https://static.igem.org/mediawiki/2019/thumb/b/b4/T--AHUT_China--E_W1_1.png/800px-T--AHUT_China--E_W1_1.png)
  ![E_W1_02](https://static.igem.org/mediawiki/2019/thumb/a/a3/T--AHUT_China--E_W1_2.png/800px-T--AHUT_China--E_W1_2.png)
  ![E_W1_03](https://static.igem.org/mediawiki/2019/thumb/e/eb/T--AHUT_China--E_W1_3.png/800px-T--AHUT_China--E_W1_3.png)
* # WEEK 2

  The measurement committee responded to the diagnostic report of the measurement results and
  confirmed that our standard curve can be used to measure the next experimental data;
  This week, the coding sequence of CA2(L203K)-N-LCTPSR([BBa\_K2949012](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2949012)) and
  CA2(L203K)-C-LCTPSR([BBa\_K2949013](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2949013)) were synthesized and cloned into the expression vector
  pET-30a(+).
* # 7.22

  The primers designed by us began to be amplified by PCR to obtain the mutant CA2
  gene;
  Agarose gel electrophoresis;

  ### Diluting All Primers

  | Rweagent | Volume of ddH2O |
  | --- | --- |
  | aldcF | 390μLddH2O |
  | aldcR | 185μLddH2O |
  | aldnF | 210μLddH2O |
  | aldnR | 285μLddH2O |

  ### Ald(c) PCR system

  | Reagent | Volume |
  | --- | --- |
  | 5X Primer STAR Buffer | 10μL |
  | dNTP Mixture(2.5mM each) | 4μL |
  | Primer 1[aldcF] | 1μL |
  | Primer 1[aldcR] | 1μL |
  | Template | 1μL |
  | Primer STAR | 0.5μL |
  | ddH2O | 32.5μL |

  ### Ald(n) PCR system

  | Reagent | Volume |
  | --- | --- |
  | 5X Primer STAR Buffer | 10μL |
  | dNTP Mixture(2.5mM each) | 4μL |
  | Primer 1[aldcF] | 1μL |
  | Primer 1[aldcR] | 1μL |
  | Template | 1μL |
  | Primer STAR | 0.5μL |
  | ddH2O | 32.5μL |

  At the end of experiment, the product of PCR was recovered by gel recovery;

  ### Recycling gel (30mL; 1% agarose gel).

  | reagent | volume |
  | --- | --- |
  | M(DL15000) | 1.5μL |
  | N1 | 50μL+5μL loading(10x) |
  | N1' | 50μL+5μL loading(10x) |
  | C1 | 50μL+5μL loading(10x) |
  | C1' | 50μL+5μL loading(10x) |

  ### Identifiying gel (20mL;1％agarose gel)

  | Reagent | Volume |
  | --- | --- |
  | M(DL15000) | 1.5μL |
  | n | 2μL+0.5μL loading(10x) |
  | c | 2μL+0.5μL loading(10x) |

  ### Electrophoresis results

  ![E_W2_01](https://static.igem.org/mediawiki/2019/3/32/T--AHUT_China--E_W2_01.png)

  M :1.5μL DL15000 DNA marker
  ①: the CA2(L203K)-N-LCTPSR mutant gene
  ②: the CA2(L203K)-C-LCTPSR mutant gene
  The experiment supplies are sterilized to ensure that the experiment is not contaminated by
  bacteria and the experimental results
  are not affected.
* # 7.23

  Transform
  pET-30a(+) (E.coli BL21); pET-28a(+) (E.coli BL21).
* # 7.24

  Pick Monoclone(4mL non-resistant LB medium and 4μL kanamycin);
* # 7.25

  The extraction and purification of recombinant plasmid;
  Measuring the Absorbance of PlasmidpET300;（5μLplasmid+55μLwater）

  | Plasmid | Absorbance | Potency |
  | --- | --- | --- |
  | pET-28a(+) | A260:A280=2.0978 | c=40μg/mL |
  | pET-30a(+) | A260:A280=2.1711 | c=80μg/mL |

  The identification of empty plasmid by electrophoresis;
  Sample: 2μL DL15000mark 1μL plasmid 1μL loading buffer.
  ![E_W2_01](https://static.igem.org/mediawiki/2019/d/d0/T--AHUT_China--E_W2_1.png)
  Lane ①: pET-28a(+)
  Lane ②: pET-30a(+)
  Lane ③: pET-28a(+)
  Lane ④: pET-30a(+)
  The CA2(L203K)-C-LCTPSR and CA2(L203K)-N-LCTPSR recombinant plasmid were constructed by double
  enzyme digestion.

  ### Empty Plasmid Enzyme Digestion System(30μL)

  | reagent | volume |
  | --- | --- |
  | NedⅠ | 1.2μL |
  | Hind Ⅲ | 1.2μL |
  | NEB Buffer 2.1 | 3μL |
  | pET-30a(+) | 1.5μg(10μL) |
  | ddH2O | 14.6μL |

  ### CA2(L203K)-C-LCTPSR Gene Enzyme Digestion System(30μL)

  | reagent | volume |
  | --- | --- |
  | NedⅠ | 1.2μL |
  | Hind Ⅲ | 1.2μL |
  | NEB Buffer 2.1 | 3μL |
  | CA2(L203K)-C-LCTPSR gene | 20μL |
  | ddH2O | 4.6μL |

  ### CA2(L203K)-N-LCTPSR Gene Enzyme Digestion System(30μL)

  | reagent | volume |
  | --- | --- |
  | NedⅠ | 1.2μL |
  | Hind Ⅲ | 1.2μL |
  | NEB Buffer 2.1 | 3μL |
  | CA2(L203K)-N-LCTPSR gene | 20μL |
  | ddH2O | 4.6μL |
* # 7.26

  ### Gel recovery

  ### Recycling gel (30mL;1％agarose gel)

  | reagent | volume |
  | --- | --- |
  | DL15000 | 2μL |
  | c | 30μL |
  | n | 30μL |
  | pET-30a(+) | 30μL |

  ### Identifying gel

  | reagent | volume |
  | --- | --- |
  | DL15000 | 2μL |
  | c | 2μL |
  | n | 2μL |
  | pET-30a(+) | 2μL |

  ### Gel electrophoresis identification results：

  ![E_W2_02](https://static.igem.org/mediawiki/2019/0/0b/T--AHUT_China--E_W2_2.png)
  Lane ①:CA2(L203K)-C-LCTPSR target gene fragment
  Lane ②:CA2(L203K)-N-LCTPSR target gene fragment
  Lane ③:Empty plasmid after enzyme digestion
  Construction of recombinant plasmid by double digestion ligation;

  ### CA2(L203K)-C-LCTPSR recombinant plasmid junction system(15μL)

  | reagent | volume |
  | --- | --- |
  | T4 connective buffer | 1.5μL |
  | Empty load after enzyme digestion[pET-30a(+)] | 6μL |
  | Target gene after enzyme digestion(C) | 2μL |
  | T4 DNA liganse | 1.5μL |
  | ddH2O | 4μL |

  ### CA2(L203K)-N-LCTPSR recombinant plasmid junction system(15μL)

  | reagent | volume |
  | --- | --- |
  | T4 connective buffer | 1.5μL |
  | Empty load after enzyme digestion[pET-30a(+)] | 6μL |
  | Target gene after enzyme digestion(N) | 2μL |
  | T4 DNA liganse | 1.5μL |
  | ddH2O | 4μL |
* # WEEK 3

  This week, we mainly constructed and identified the recombinant plasmid by restriction enzyme
  digestion and sequencing.
  Transformation;
  pET-30a(+)-CA2(L203K)-C-LCTPSR (E.coli BL21);
  pET-30a(+)-CA2(L203K)-N-LCTPSR (E.coli BL21)
  Pick Monoclone(4mL non-resistant LB medium and 4μL kanamycin):

  ![E_W3_01_C-terminal](https://static.igem.org/mediawiki/2019/thumb/9/9c/T--AHUT_China--E_W3_1.jpg/450px-T--AHUT_China--E_W3_1.jpg)
  ![E_w3_02_N-terminal](https://static.igem.org/mediawiki/2019/thumb/1/1d/T--AHUT_China--E_W3_2.jpg/449px-T--AHUT_China--E_W3_2.jpg)
  (pET-30a(+)-CA2(L203K)-C-LCTPSR E.coli BL21 colony)
  (pET-30a(+)-CA2(L203K)-N-LCTPSR E.coli BL21 colony)
  The extraction and purification of recombinant plasmid；
  Identification of recombinant plasmids by gel electrophoresis；
  ![E_w3_03](https://static.igem.org/mediawiki/2019/5/55/T--AHUT_China--E_W3_3.png)
  LaneM:2μL DL15000 DNA marker
  Lane① and ②: CA2(L203K)-C-LCTPSR target gene(no results）
  Lane③ and ④: CA2(L203K)-N-LCTPSR target gene(the arrow indicated)
  The identification of c-terminal recombinant plasmid by restriction enzyme digestion showed no
  results.

  ### The identificiation of CA2(L203K)-N-LCTPSR recombinant plasmid by restriction enzyme digestion.

  ### Enzyme digestion system of pET-30a(+)-CA2(L203K)-N-LCTPSR

  | reagent | volume |
  | --- | --- |
  | pET-30a(+)- CA2(L203K)-N-LCTPSR | 5μL |
  | NedⅠ | 1.2μL |
  | Hind Ⅲ | 1.2μL |
  | NEB Buffer 2.1 | 3μL |
  | ddH2O | 9.6μL |

  ### Identifying gel

  | reagent | volume |
  | --- | --- |
  | DL15000 | 2μL |
  | pET-30a(+)-CA2(L203K)-N-LCTPSR before enzyme digestion | 2μL |
  | pET-30a(+)-CA2(L203K)-N-LCTPSR after enzyme digestion | 10μL |

  Agarose Gel Electrophoresis of CA2(L203K)-N-LCTPSR recombinant plasmid and its identification by
  enzyme digestion (NdeⅠand Hind Ⅲ);
  ![E_w3_04](https://static.igem.org/mediawiki/2019/6/6e/T--AHUT_China--E_W3_41.jpg)
  Lane M: DL15000 marker;
  Lane 1: CA2(L203K)-N-LCTPSR recombinant plasmid;
  Lane 2: Enzyme digestion band of CA2(L203K)-N-LCTPSR, the length was 834 bp (the arrow
  indicated)
* # 7.29

  Since the identification of CA2(L203K)-C-LCTPSR recombinant plasmid was not successful, the transformed
  recombinant plasmid was used to re-select monoclones.
* # 7.30

  No result was found by gel electrophoresis.
* # 7.31

  For the second time, E. coli BL21 was used for transformation, but no colonies were found.
* # 8.1

  Start the third construction of CA2(L203K)-C-LCTPSR recombinant plasmid ;
  Double digestion connection and transformation.

  ### Connecting System（15μL）

  | reagent | volume |
  | --- | --- |
  | T4 connective buffer | 1.5μL |
  | Empty load after enzyme digestion[pET-30a(+)] | 6μL |
  | CA2(L203K)-C-LCTPSR recombinant plasmid after enzyme digestion | 2μL |
  | T4 DNA liganse | 1.5μL |
  | ddH2O | 4μL |
* # 8.2

  Pick monoclone;
  Redo CA2(L203K)-C-LCTPSR gene fragments in order to avoid further failure;

  ### pET-30a(+) Enzyme Digestion System(30μL)

  | reagent | volume |
  | --- | --- |
  | NedⅠ | 1.2μL |
  | Hind Ⅲ | 1.2μL |
  | NEB Buffer 2.1 | 3μL |
  | pET-30a(+) | 1.5μg |
  | ddH2O | 4.6μL |

  ### Gel recovery；1％agarose gel;30mL；

  ### Identifying gel ;1％agarose gel;20mL;

  | reagent | volume |
  | --- | --- |
  | DL15000 | 2μL |
  | CA2(L203K)-C-LCTPSR | 2μL |
  | CA2(L203K)-N-LCTPSR | 2μL |
  | pET-30a(+) | 2μL |

  ### Identification results of double digestion

  ![E_w3_05](https://static.igem.org/mediawiki/2019/8/83/T--AHUT_China--E_W3_5.png)

  ①:CA2(L203K)-N-LCTPSR target gene fragment
  ②:CA2(L203K)-C-LCTPSR target gene fragment
  ③:Empty plasmid after the enzyme digestion
* # 8.3

  CA2(L203K)-C-LCTPSR recombinant plasmid was identified by agarose gel electrophoresis.
  ![E_w3_06](https://static.igem.org/mediawiki/2019/1/19/T--AHUT_China--E_W3_6.jpg)
  ①②③ are CA2(L203K)-C-LCTPSR recombinant plasmid(the arrow indicated)
  superhelix occurred in plasmid ②

  The identificiation of CA2(L203K)-C-LCTPSR recombinant plasmid by restriction enzyme digestion.

  ### Enzyme digestion system

  | Ingredient | Dosage |
  | --- | --- |
  | NedⅠ | 1.2μL |
  | Hind Ⅲ | 1.2μL |
  | NEB Buffer 2.1 | 3μL |
  | C1/C2/C4 | 5μL |
  | ddH2O | 9.6μL |

  ### Agarose gel electrophoresis of CA2(L203K)-C-LCTPSR recombinant plasmid and its identification by enzyme digestion (NedⅠand Hind Ⅲ);

  ![E_w3_07](https://static.igem.org/mediawiki/2019/1/1f/T--AHUT_China--E_W3_7.jpg)
  Lane M: DL15000 marker;
  Lane 1: CA2(L203K)-C-LCTPSR recombinant plasmid;
  Lane 2: Enzyme digestion band of CA2(L203K)-C-LCTPSR,the length was 834 bp (the arrow
  indicated).
* # 8.4

  Sequencing results：
  CA2(L203K)-N-LCTPSR and CA2(L203K)-C-LCTPSR gene sequencing is correct
  ![E_w3_08](https://static.igem.org/mediawiki/2019/thumb/4/4a/T--AHUT_China--E_W3_8.png/800px-T--AHUT_China--E_W3_8.png)
  ![E_w3_09](https://static.igem.org/mediawiki/2019/thumb/d/db/T--AHUT_China--E_W3_9.png/800px-T--AHUT_China--E_W3_9.png)
* # WEEK 4

  This week,IPTG was used to include CA2(L203k)-N-LCTPSR and CA2(L203K)-C-LCTPSR expression in
  recombinant E.coli BL21 (DE3)and SDS-PAGE and Western Blot were used to identify the proteins.
  At the same time,
  experiments were carried out in cooperation with Ocean University of China.
* # 8.6

  Pick monoclones
  [pET-30a(+)-CA2(L203K)-C-LCTPSR(E.coli BL21) and pET-30a(+)-CA2(L203K)-N-LCTPSR(E.coli BL21)];(4mL non-resistant LB medium and 4 μ L kanamycin);
  Expand culture (enrich bacterial solution).
* # 8.7

  SDS-PAGE detection of CA2(L203K)-N-LCTPSR and CA2(L203K)-C-LCTPSR protein expression;

  ### Preparation of separation gel（15% 10mL）

  | Reagent | Volume |
  | --- | --- |
  | distilled water | 2.3mL |
  | Acr-Bis | 5mL |
  | Tris-hcl(pH=8.8) | 2.5mL |
  | 10%SDS | 0.1mL |
  | 10%Ammonium persulfate | 0.1mL |
  | TEMED | 0.008mL |

  ### Preparation of concentrated gel（5% 4mL）

  | Reagent | Volume |
  | --- | --- |
  | distilled water | 2.7mL |
  | Acr-Bis | 0.67mL |
  | Tris-hcl(pH=6.8) | 0.5mL |
  | 10%SDS | 0.04mL |
  | 10%Ammonium persulfate | 0.04mL |
  | TEMED | 0.004mL |
* # 8.8

  ### Electrophoretic Sample

  | Reagent | Volume |
  | --- | --- |
  | mark | 1.5μL |
  | 1xSDS loading Buffer | 20μL |
  | n(-) | 15μL |
  | n(+) | 15μL |
  | c(-) | 15μL |
  | c(+) | 15μL |
  | 1×SDS loading Buffer 15μL | 15μL |

### Testing

Coomassie brilliant blue staining for 20 minutes
No target protein bands were found after decolorization.

### First redo

The monoclones of pET-30a(+)-CA2(L203K)-C-LCTPSR(BL21) were selected, and did not grow and the colony died due to the underlined culture, so it needed to be re-transformed and cultured.

- # 8.9

  After expanding culture, IPTG induction and SDS-PAGE detection,no significant protein expression
  was found.

  The experimental cooperation with Ocean University of China has been started.
  The operation is as follows:
  Adda solid puncture bacteria (chassis: Escherichia coli DH5alphaZ1) were collected and
  concentrated in liquid culture. Single colonies were separated by lines after 10 hours.
  Monoclones were selected after 20 hours. After 12 hours, different media were added to induce
  culture for 2 hours, 4 hours, 6 hours, 8 hours, 10 hours and 12 hours, and then the fluorescence
  and OD600 were measured respectively.
  The measurement results are as follows:
  ![E_w3_091](https://static.igem.org/mediawiki/2019/thumb/5/58/T--AHUT_China--E_W14_1.png/800px-T--AHUT_China--E_W14_1.png)
- # 8.10

  Transform pET-30a(+)-CA2(L203K)-N-LCTPSR (BL21)(correctly sequenced plasmid)
  Pick Monoclonal; pET-30a(+)-CA2(L203K)-C-LCTPSR (BL21)
- # 8.11

  The transformation of pET-30a(+)-CA2(L203K)-N-LCTPSR(BL21) was successful.
  pET-30a(+)-CA2(L203K)-C-LCTPSR(BL21) single clone grows colony, continued to expand
  the culture for 4 hours, induced for 4 hours, SDS-PAGE detection protein, but did not express
  successfully.
- # WEEK 5

  Because no results of the CA2(L203K)-C-LCTPSR(BL21) protein expression was detected, the
  experiment continued,we switched to E. coli TB1 for transformation,fortunately, both of
  CA2(L203K)-N-LCTPSR protein and CA2(L203K)-C-LCTPSR protein could be successfully expressed in
  TB1 strain after SDS-PAGE. And we transformed P-BAD-FGE and pET-30a(+)-CA2(L203K)-C-LCTPSR
  plasmid into E.coli TB1
- # 8.12

  Expanded culture of pET-30a(+)-CA2(L203K)-C-LCTPSR (BL21), IPTG induction, sample preparation;
  Transform pET-30a(+)-CA2(L203K)-C-LCTPSR(TB1) and pET-30a(+)-CA2(L203K)-N-LCTPSR (TB1);
- # 8.13

  1.Transform result:
  ![E_W5_01](https://static.igem.org/mediawiki/2019/thumb/7/79/T--AHUT_China--E_W5-1.jpg/799px-T--AHUT_China--E_W5-1.jpg)
  ![E_w5_02_](https://static.igem.org/mediawiki/2019/thumb/c/c2/T--AHUT_China--E_W5_2.jpg/799px-T--AHUT_China--E_W5_2.jpg)
  [pET-30a(+)-CA2(L203K)-C-LCTPSR(TB1)]
  [pET-30a(+)-CA2(L203K)-N-LCTPSR(TB1)]
  2. Pick monoclonal
  pET-30a(+)-CA2(L203K)-C-LCTPSR(TB1)and pET-30a(+)-CA2(L203K)-N-LCTPSR(TB1)(4mL non-resistant LB medium and 4 μ L kanamycin).
- # 8.14

  Expand culture; IPTG induction; Sample preparation; Electrophoretic dyeing and decolorization;
  The results are as follows:
  ![E_W5_03](https://static.igem.org/mediawiki/2019/thumb/f/f2/T--AHUT_China--E_W5_3.png/465px-T--AHUT_China--E_W5_3.png)
  ![E_w5_04_](https://static.igem.org/mediawiki/2019/thumb/8/83/T--AHUT_China--E_W5_4.png/482px-T--AHUT_China--E_W5_4.png)
  Fig.1 SDS-PAGE analysis for CA2(L203K)-C-LCTPSR cloned in pET-30a(+) vector and expressed in
  E.coli TB1
  Fig.2 SDS-PAGE analysis for CA2(L203K)-N-LCTPSR cloned in pET-30a(+) vector and expressed in
  E.coli TB1
- # 8.15

  Pick monoclonal;
- # 8.16

  All monoclonal colonies grow.
  Preserve bacteria [pET-30a(+)-CA2(L203K)-C-LCTPSR(TB1), pET-30a(+)-CA2(L203K)-N-LCTPSR(TB1) and
  pET30a-CcaA(BL21)](-80℃)
- # 8.17

  1.Plasmid extraction of pET-30a(+)-CA2(L203K)-C-LCTPSR (TB1)
  2.Plasmid co-transformation;[pBAD-FGE+pET-30a(+)-CA2(L203K)-N-LCTPSR]
- # 8.18

  Pick monoclonal(plasmid co-transformed bacteria) [LB Medium (+1mL AMP and KANA)]
- # WEEK 6

  This week, the pET-30a(+)-CA2(L203K)-C-LCTPSR protein was purified and concentrated.
- # 8.19

  Preparation of KANA and IPTG, Arabic Sugar Solution;
  Sterilization by 0.22 μm membrane;
  Expand culture E.coli TB1[2mL Bacterial Solution + 200mL LB Medium (+1mL AMP, KANA)];
  Measure OD600 with 200μL;
  The result of measuring OD600 is 0.641;
  Induced expression with Arabic Sugar Solution, 30 minutes later + IPTG
- # 8.20

  Cell lysis to obtain protein;
  Centrifugal ultrasonic comminution of cells.
- # 8.21

  Preparation of 250X, 50X, 5X imidazole 1000 mL;
  Adjust PBS to pH 7.4;
  Purification of Nickel Column Protein.
- # 8.23

  Protein concentrate was centrifuged at 4000rpm for 20 min at 4 ℃.
- # WEEK 7

  This week, we completed the experiments of pET-30a(+)-CA2(L203K)-N-LCTPSR co-transformation,
  purification and concentration of expressed proteins, and the related experiments of validating
  part.
- # 8.26

  The concentrated protein was identified by dyeing, and the protein was separated.
- # 8.28

  Start characterizing the existing biobrick part BBa\_K2762004 (CcaA)
  Pick monoclones (BL21-CcaA)
- # 8.29

  1. Make double-resistance medium of KANA resistance + AMP resistance.
  2. Expand BL21-CcaA culture, adding 4ml bacterial solution to 200 ml kanamycin culture
  medium (100μl), putting it in shaking table, 120rpm, 37℃ for 4 hours, and adding 1 ml IPTG to
  induce expression.
  3. Plasmid co-transformation TB1 [P-BAD-FGE+pET-30a(+)-CA2(L203K)-N-LCTPSR]
  4. SDS-PAGE analysis for CcaA cloned in pET-30a(+) vector and expressed in BL21 strain
  The results showed that pET-30a(+)-CcaA could be successfully expressed in BL21 strain.
  ![E_W5_03](https://static.igem.org/mediawiki/2019/thumb/4/48/T--AHUT_China--E_W5_5.png/508px-T--AHUT_China--E_W5_5.png)
- # 8.30

  1. Centrifugal bacterial fluid (BL21-CcaA)
  2. Broken strain (BL21-CcaA)
  3. Pick monoclones (CA2(L203K)-N-LCTPSR-CHO-FGE)
- # 8.31

  1. Expand culture of E.coli TB1 [4ml bacterium solution + 200mL LB(100μLKANA + 100μLAMP+)]
  2. Protein Purification [Nickel Column Purification]
  3. Protein Concentration
  4. Induced expression [+1 ml arabinose ; +1 ml IPTG after 30 min]
- # WEEK 8

  In order to get more CA2(L203K)-N-LCTPSR and CA2(L203K)-C-LCTPSR protein,we continue to do protein purification and concentration (CA2(L203K)-N-LCTPSR-FGE) this week.
  Resuspension, crushing strains, protein purification and centrifugal concentration.
- # WEEK 9

  This week, CA2(L203K)-N-LCTPSR and CA2(L203K)-C-LCTPSR protein immobilization and esterase
  activity assay were completed.
- # 9.10

  Immobilization;

  ### Standard protein concentration determination.

  ![E_w9_01_](https://static.igem.org/mediawiki/2019/thumb/1/1c/T--AHUT_China--E_W9_11.png/800px-T--AHUT_China--E_W9_11.png)

  Standard protein curve
  ![E_w9_01_](https://static.igem.org/mediawiki/2019/8/8c/T--AHUT_China--E_W9_1.png)

  | Sample | Absorbance | Average | After deducting PBS |
  | --- | --- | --- | --- |
  | CA2(L203K)-C-LCTPSR immobilization (dilution 10 times) | 0.401 0.379 － | 0.39 | 0.103 |
  | CA2(L203K)-C-LCTPSR not immobilized (diluted 10 times) | 0.414 0.41 － | 0.412 | 0.125 |
  | CA2(L203K)-N-LCTPSR immobilization (dilution 10 times) | 0.412 0.416 0.404 | 0.410667 | 0.123667 |
  | CA2(L203K)-N-LCTPSR not immobilized (diluted 10 times) | － 0.436 0.43 | 0.433 | 0.146 |
  | CA2(L203K)-C-LCTPSR (diluted 10 times) | 0.431 0.431 0.432 | 0.431333 | 0.144333 |
  | CA2(L203K)-N-LCTPSR (diluted 10 times) | 0.456 0.442 0.456/td> 0.451333 | 0.164333 | |
  | PBS | 0.292 0.284 0.285 | 0.287 | 0 |

  It can be obtained from standard protein curve y=1.0633x+0.0386.

  | CA2(L203K)-C-LCTPSR immobilization (dilution 10 times)= 0.0605662 mg/ml | 39.09% |
  | --- | --- |
  | CA2(L203K)-C-LCTPSR not immobilized (diluted 10 times)= 0.0812565 mg/ml | 18.28% |
  | CA2(L203K)-C-LCTPSR (diluted 10 times)= 0.0994385 mg/ml | 20.81% |
  | CA2(L203K)-N-LCTPSR immobilization (dilution 10 times)= 0.0800000 mg/ml | 32.35% |
  | CA2(L203K)-N-LCTPSR not immobilized (diluted 10 times)= 0.1010063 mg/ml | 14.58% |
  | CA2(L203K)-N-LCTPSR (diluted 10 times)= 0.1182479 mg/ml | 17.77% |
- # 9.12

  Esterase assay for enzyme activity

  ### Preparation of Standard BSA Solution

  ![E_w9_02_](https://static.igem.org/mediawiki/2019/a/a2/T--AHUT_China--E_W9_2.png)
  ![E_w9_03_](https://static.igem.org/mediawiki/2019/thumb/4/41/T--AHUT_China--E_W9_3.png/800px-T--AHUT_China--E_W9_3.png)
  ![E_w9_04_](https://static.igem.org/mediawiki/2019/thumb/8/88/T--AHUT_China--E_W9_4.png/800px-T--AHUT_China--E_W9_4.png)
  ![E_w9_06_](https://static.igem.org/mediawiki/2019/0/0e/T--AHUT_China--E_W9_6.png)
  ![E_w9_07_](https://static.igem.org/mediawiki/2019/thumb/6/6d/T--AHUT_China--E_W9_7.png/1176px-T--AHUT_China--E_W9_7.png)
- # WEEK 10

  In order to obtain more proteins for enzyme immobilization and multiple assay of enzyme
  activity,this week we successfully re-transformed pET-30a(+)-CA2(L203K)-C-LCTPSR,
  pET-30a(+)-CA2(L203K)-N-LCTPSR.And completed the experiments of purification and concentration
  of expressed proteins.
- # 9.13

  Make double-resistance medium of KANA resistance + AMP resistance.
- # 9.14

  Transformation;
  pET-30a(+)-CA2(L203K)-C-LCTPSR (E.coli TB1);
  pET-30a(+)-CA2(L203K)-N-LCTPSR (E.coli TB1).
- # 9.15

  1.Expanded culture of E.coli TB1 [4ml bacterium solution + 200mL LB(100μLKANA + 100μLAMP+)]
  2.Induced expression[+1 ml arabinose ; +1ml IPTG after 30min];
  pET-30a(+)-CA2(L203K)-C-LCTPSR (E.coli TB1)
  pET-30a(+)-CA2(L203K)-N-LCTPSR (E.coli TB1)；
- # 9.16

  1.Protein Purification [Nickel Column Purification]
  2.Protein Concentration
- # 9.17

  SDS-PAGE detection
  ![E_w10_01_](https://static.igem.org/mediawiki/2019/e/eb/T--AHUT_China--E_W10_1.jpg)
  SDS-PAGE analysis of purified CA2(L203K)-N-LCTPSR protein
  ![E_w10_02_](https://static.igem.org/mediawiki/2019/d/dc/T--AHUT_China--E_W10_2.jpg)
  SDS-PAGE of purified CA2(L203K)-C-LCTPSR protein
- # WEEK 11

  This week,CA2(L203K)-C-LCTPSR protein and CA2(L203K)-N-LCTPSR immobilization and esterase assay
  were completed.
- # 9.22

  Immobilized CA2(L203K)-C-LCTPSR protein and CA2(L203K)-N-LCTPSR protein.
- # 9.24

  Esterase assay for enzyme activity of free CA2(L203K) protein, free and immobilized
  CA2(L203K)-C-LCTPSR protein, free and immobilizedCA2(L203K)-N-LCTPSR protein at 37℃ and 50℃.
  The results showed as follows:
  ![E_w10_02_](https://static.igem.org/mediawiki/2019/thumb/4/41/T--AHUT_China--E_W11_1.png/784px-T--AHUT_China--E_W11_1.png)
  Esterase activity analysis of free CA2(L203K) and CA2(L203K)-C-LCTPSR protein and immobilized
  CA2(L203K)-C-LCTPSR protein at 37℃
  ![E_w10_02_](https://static.igem.org/mediawiki/2019/thumb/7/7a/T--AHUT_China--E_W12_1.png/784px-T--AHUT_China--E_W12_1.png)
  Esterase activity analysis of free CA2(L203K) and CA2(L203K)-C-LCTPSR protein and immobilized
  CA2(L203K)-C-LCTPSR protein at 50℃
  ![E_w10_02_](https://static.igem.org/mediawiki/2019/thumb/f/f0/T--AHUT_China--E_W11_3.png/784px-T--AHUT_China--E_W11_3.png)
  Esterase activity analysis of free CA2(L203K)-N-LCTPSR protein and immobilized
  CA2(L203K)-N-LCTPSR protein at 37℃ and 50℃
- # WEEK 12

  This week, the reuse ability of CO2 capture of the immobilized CA2(L203K)-C-LCTPSR was tested by our designed simulation model.
  The result as follows:
  ![E_w10_02_](https://static.igem.org/mediawiki/2019/thumb/7/7f/T--AHUT_China--E_W11_5.png/783px-T--AHUT_China--E_W11_5.png)

![](https://static.igem.org/mediawiki/2019/thumb/8/8e/T--AHUT_China--dnat.png/234px-T--AHUT_China--dnat.png)

# CATALOGUE

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CONTACT US

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# Team:AHUT China/Experiments

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[![](https://placehold.it/350x150/ececec/cecece)](https://2019.igem.org/Team%3AAHUT_China)
[HOME](https://2019.igem.org/Team%3AAHUT_China)

一级目录 TEAM

[二级目录1 Team Members](https://2019.igem.org/Team%3AAHUT_China/Team)
[二级目录2 Collaborations](https://2019.igem.org/Team%3AAHUT_China/Collaborations)

一级目录 PROJECT

[二级目录1 Description](https://2019.igem.org/Team%3AAHUT_China/Description)
[二级目录2 Design](https://2019.igem.org/Team%3AAHUT_China/Design)
[二级目录3 Experiments](https://2019.igem.org/Team%3AAHUT_China/Experiments)
[Notebook](https://2019.igem.org/Team%3AAHUT_China/Notebook)
[Contribution](https://2019.igem.org/Team%3AAHUT_China/Contribution)
[Results](https://2019.igem.org/Team%3AAHUT_China/Results)
[Demonstrate](https://2019.igem.org/Team%3AAHUT_China/Demonstrate)
[Improve](https://2019.igem.org/Team%3AAHUT_China/Improve)
[Attributions](https://2019.igem.org/Team%3AAHUT_China/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AAHUT_China/Parts)
[Basic Parts](https://2019.igem.org/Team%3AAHUT_China/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AAHUT_China/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3AAHUT_China/Part_Collection)

[SAFETY](https://2019.igem.org/Team%3AAHUT_China/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3AAHUT_China/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3AAHUT_China/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3AAHUT_China/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3AAHUT_China/Hardware)
[Measurement](https://2019.igem.org/Team%3AAHUT_China/Measurement)
[Model](https://2019.igem.org/Team%3AAHUT_China/Model)
[Plant](https://2019.igem.org/Team%3AAHUT_China/Plant)
[Software](https://2019.igem.org/Team%3AAHUT_China/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=AHUT_China)

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:AHUT China/Results

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![](https://static.igem.org/mediawiki/2019/e/ee/2019_team_menu_icon.svg)

[![](https://placehold.it/350x150/ececec/cecece)](https://2019.igem.org/Team%3AAHUT_China)
[HOME](https://2019.igem.org/Team%3AAHUT_China)

一级目录 TEAM

[二级目录1 Team Members](https://2019.igem.org/Team%3AAHUT_China/Team)
[二级目录2 Collaborations](https://2019.igem.org/Team%3AAHUT_China/Collaborations)

一级目录 PROJECT

[二级目录1 Description](https://2019.igem.org/Team%3AAHUT_China/Description)
[二级目录2 Design](https://2019.igem.org/Team%3AAHUT_China/Design)
[二级目录3 Experiments](https://2019.igem.org/Team%3AAHUT_China/Experiments)
[Notebook](https://2019.igem.org/Team%3AAHUT_China/Notebook)
[Contribution](https://2019.igem.org/Team%3AAHUT_China/Contribution)
[Results](https://2019.igem.org/Team%3AAHUT_China/Results)
[Demonstrate](https://2019.igem.org/Team%3AAHUT_China/Demonstrate)
[Improve](https://2019.igem.org/Team%3AAHUT_China/Improve)
[Attributions](https://2019.igem.org/Team%3AAHUT_China/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AAHUT_China/Parts)
[Basic Parts](https://2019.igem.org/Team%3AAHUT_China/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AAHUT_China/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3AAHUT_China/Part_Collection)

[SAFETY](https://2019.igem.org/Team%3AAHUT_China/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3AAHUT_China/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3AAHUT_China/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3AAHUT_China/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3AAHUT_China/Hardware)
[Measurement](https://2019.igem.org/Team%3AAHUT_China/Measurement)
[Model](https://2019.igem.org/Team%3AAHUT_China/Model)
[Plant](https://2019.igem.org/Team%3AAHUT_China/Plant)
[Software](https://2019.igem.org/Team%3AAHUT_China/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=AHUT_China)

# Results

Here you can describe the results of your project and your future plans.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the part's Main Page on the Registry.** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)