iGEM 2016 — Team HUST-China

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# Team:HUST-China/Notebook

Notebook

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* [HOME](https://2016.igem.org/Team%3AHUST-China)
* PROJECT
  + [Background](https://2016.igem.org/Team%3AHUST-China/project/background)
  + [Description](https://2016.igem.org/Team%3AHUST-China/Description)
  + [Proof of concept](https://2016.igem.org/Team%3AHUST-China/Proof)
  + [Application](https://2016.igem.org/Team%3AHUST-China/Demonstrate)
  + [Previous improvement](https://2016.igem.org/Team%3AHUST-China/project/inprovement)
  + [Safety](https://2016.igem.org/Team%3AHUST-China/Safety)
* WETLAB
  + [Experiments](https://2016.igem.org/Team%3AHUST-China/Experiments)
  + [Results](https://2016.igem.org/Team%3AHUST-China/Results)
  + [Interlab](https://2016.igem.org/Team%3AHUST-China/InterLab)
  + [Notebook](https://2016.igem.org/Team%3AHUST-China/Notebook)
  + [Protocols](https://2016.igem.org/Team%3AHUST-China/wetlab/protocols)
* MODELING
  + [Overview](https://2016.igem.org/Team%3AHUST-China/Model)
  + [Prokaryotic circuit](https://2016.igem.org/Team%3AHUST-China/Model/model-pro)
  + [Eukaryotic circuit](https://2016.igem.org/Team%3AHUST-China/Model/model-euk)
  + [Application circuit](https://2016.igem.org/Team%3AHUST-China/Model/model-app)
* PARTS
  + [Summary](https://2016.igem.org/Team%3AHUST-China/Parts)
  + [Basic part](https://2016.igem.org/Team%3AHUST-China/Basic_Part)
  + [Composite part](https://2016.igem.org/Team%3AHUST-China/Composite_Part)
  + [Part Collection](https://2016.igem.org/Team%3AHUST-China/Part_Collection)
* HUMAN PRACTICES
  + [Integrated HP](https://2016.igem.org/Team%3AHUST-China/Integrated_Practices)
  + [Collaborations](https://2016.igem.org/Team%3AHUST-China/Collaborations)
* TEAM
  + [Team Roster](https://2016.igem.org/Team%3AHUST-China/Team)
  + [Attributions](https://2016.igem.org/Team%3AHUST-China/Attributions)

## Notebook

#### see how these parts were constructed？

## CII

###### 7.24

###### PCR

###### Yufeng Mo

|  |  |
| --- | --- |
| Name Total | |
| CII 1 50 μl | |
| CII 2 50 μl | |
| CII control group 30 μl | |

###### 7.25

###### Restriction Enzyme Digestion

###### Yufeng Mo

|  |  |
| --- | --- |
| Name Enzyme | |
| CII XbaI and PstI | |

###### 7.26

###### DNA ligation&transformation

###### Yufeng Mo

|  |  |  |  |
| --- | --- | --- | --- |
| Name | Backbone | Strain | Medium |
| CII | psb1c3(Xba I and Pst I cut) | DH5α | LB(chl added) |

###### 7.27

###### Colony PCR

###### Yufeng Mo

|  |  |
| --- | --- |
| Number | Name |
| 1 | CII |

![](https://static.igem.org/mediawiki/2016/f/f8/T--HUST-China--notebook-CII-fig01.png)

## CIII

###### 7.14

###### PCR

###### Yufeng Mo

|  |  |
| --- | --- |
| Name Total | |
| CIII 1 50 μl | |

###### 7.15

###### Restriction Enzyme Digestion

###### Yufeng Mo

|  |  |
| --- | --- |
| Name Enzyme | |
| plasmid psb1c3 1 XbaI and SpeI | |
| CIII XbaI and SpeI | |

![](https://static.igem.org/mediawiki/2016/8/8c/T--HUST-China--notebook-CIII-fig01.png)

###### 7.15

###### Dephosphorylation

###### Yufeng Mo

###### 7.17

###### DNA ligation&transformation

###### Yufeng Mo

|  |  |  |  |
| --- | --- | --- | --- |
| Name Backbone Strain Medium | | | |
| CIII psb1c3(XbaI and SpeI cut) DH5α LB(chl added) | | | |

###### 7.18

###### Colony PCR

###### Yufeng Mo

|  |  |
| --- | --- |
| Number Name | |
| 1 CIII 1 | |
| 2 CIII 2 | |
| 3 CIII 3 | |
| 4 CIII 4 | |
| 5 CIII 5 | |
| 6 CIII 6 | |

![](https://static.igem.org/mediawiki/2016/c/ca/T--HUST-China--notebook-CIII-fig02.png)

## RBS-CII

###### PCR

###### Yufeng Mo

|  |  |
| --- | --- |
| Name Total | |
| RBS-CII 2 40 μl | |
| RBS-CII 1 40 μl | |
| RBS-CII 0 20 μl | |

###### 9.4

###### Restriction Enzyme Digestion

###### Yufeng Mo

|  |  |
| --- | --- |
| Name Enzyme | |
| RBS-CII XbaI and PstI | |

###### 9.4

###### DNA ligation&transformation

###### Yufeng Mo

|  |  |  |  |
| --- | --- | --- | --- |
| Name Backbone Strain Medium | | | |
| RBS-CII psb1c3(Xba I and Pst I cut) DH5α LB(chl added) | | | |

###### 9.5

###### Colony PCR

###### Yufeng Mo

|  |  |
| --- | --- |
| Number Name | |
| 1 RBS-CII 1 | |
| 2 RBS-CII 2 | |

![](https://static.igem.org/mediawiki/2016/c/ca/T--HUST-China--notebook-RBS-CII-fig01.png)

## RBS-CIII

###### 8.7

###### PCR

###### Ziyi Jiang

|  |  |
| --- | --- |
| Name Total | |
| RBS-CIII 50 μl | |
| RBS-CIII 50 μl | |

![](https://static.igem.org/mediawiki/2016/5/58/T--HUST-China--notebook-RBS-CIII-fig01.png)

###### 8.7

###### Restriction Enzyme Digestion

###### Ziyi Jiang

|  |  |
| --- | --- |
| Name Enzyme | |
| psb1c3 XbaI and PstI | |
| RBS-CIII XbaI and PstI | |

###### 8.10

###### DNA ligation&transformation

###### Ziyi Jiang

|  |  |  |  |
| --- | --- | --- | --- |
| Name Backbone Strain Medium | | | |
| RBS-CIII psb1c3(XbaI and PstI cut) DH5α LB(chl added) | | | |

###### 8.11

###### Colony PCR

###### Ziyi Jiang

|  |  |
| --- | --- |
| Number Name | |
| 2 RBS-CIII | |

![](https://static.igem.org/mediawiki/2016/9/94/T--HUST-China--notebook-RBS-CIII-fig02.png)

## RBS-CIII-RBS-CIII

###### 8.18

###### Restriction Enzyme Digestion

###### Ziyi Jiang

|  |  |
| --- | --- |
| Name Enzyme | |
| RBS-CIII EcoRI and SpeI | |
| RBS-CIII EcoRI and XbaI | |

![](https://static.igem.org/mediawiki/2016/6/67/T--HUST-China--notebook-RBS-CIII-RBS-CIII-fig01.png)

###### 8.18

###### DNA ligation&Transformation

###### Ziyi Jiang

|  |  |  |  |
| --- | --- | --- | --- |
| Name Backbone Strain Medium | | | |
| CIII-RBS CIII-RBS-psb1c3(EcoRI and XbaI cut) DH5α LB(chl added) | | | |

###### 8.19

###### Colony PCR

###### Ziyi Jiang

|  |  |
| --- | --- |
| Number Name | |
| 13 RBS-CIII-RBS-CIII-psb1c3 | |
| 19 RBS-CIII-RBS-CIII-psb1c3 | |

![](https://static.igem.org/mediawiki/2016/6/67/T--HUST-China--notebook-RBS-CIII-RBS-CIII-fig02.png)

## RBS-CII-RBS-CII

###### 9.12

###### PCR

###### Yufeng Mo

|  |  |
| --- | --- |
| Name Total | |
| RBS-CII 50 μl | |

###### 9.14

###### Restriction Enzyme Digestion

###### Yufeng Mo

|  |  |
| --- | --- |
| Name Enzyme | |
| RBS-CII-psb1c3 EcoRI and XbaI | |
| RBS-CII-psb1c3 EcoRI and SpeI | |

###### 9.14

###### DNA ligation&transformation

###### Yufeng Mo

|  |  |  |  |
| --- | --- | --- | --- |
| Name Backbone Strain Medium | | | |
| RBS-CII RBS-CII-psb1c3(EcoRI and XbaI cut) DH5α LB(chl added) | | | |

###### 9.15

###### Colony PCR

###### Zhi Zeng

|  |  |
| --- | --- |
| Number Name | |
| 1 RBS-CII-RBS-CII-psb1c3 | |

![](https://static.igem.org/mediawiki/2016/e/e2/T--HUST-China--notebook-RBS-CII-RBS-CII-fig01.png)

## pRM-RBS-GFP-LAVtag

###### 7.18

###### PCR

###### Xiaoyang Li

|  |  |
| --- | --- |
| Name Total | |
| GFP-First 50 μl | |

|  |  |
| --- | --- |
| Name Total | |
| pRM-RBS-GFP-LAVtag 30 μl | |
| pRM-RBS-GFP-LAVtag 30 μl | |
| pRM-RBS-GFP-LAVtag 30 μl | |

![](https://static.igem.org/mediawiki/2016/b/b7/T--HUST-China--notebook-pRM-RBS-GFP-LVAtag-fig01.png)

###### 7.19

###### Restriction Enzyme Digestion

###### Xiaoyang Li

|  |  |
| --- | --- |
| Name Enzyme | |
| GFP EcoRI and SpeI | |
| psb1c3 EcoRI and SpeI | |

###### 7.20

###### DNA ligation&transformation

###### Xiaoyang Li

|  |  |  |  |
| --- | --- | --- | --- |
| Name Backbone Strain Medium | | | |
| pRM-RBS-GFP-LAVtag psb1c3(EcoRI and SpeI cut) DH5α LB(chl added) | | | |

###### 7.24

###### Colony PCR

###### Xiaoyang Li

|  |  |
| --- | --- |
| Number Name | |
| 1 pRM-RBS-GFP-LVAtag | |

![](https://static.igem.org/mediawiki/2016/e/ec/T--HUST-China--notebook-pRM-RBS-GFP-LVAtag-fig02.png)

###### 8.21

###### Restriction Enzyme Digestion

###### Yufeng Mo

|  |  |
| --- | --- |
| Name Enzyme | |
| pRM-RBS-GFP-LVAtag EcoRI and PstI | |
| pETDuet-1 EcoRI and PstI | |

###### 8.21

###### DNA ligation&transformation

###### Yufeng Mo

|  |  |  |  |
| --- | --- | --- | --- |
| Name Backbone Strain Medium | | | |
| pRM-RBS-GFP-LVAtag pETDuet-1(EcoRI and PstI cut) DH5α LB(Amp added) | | | |

###### 8.22

###### Colony PCR

###### Yufeng Mo

|  |  |
| --- | --- |
| Number Name | |
| 1 pRM-RBS-GFP-LVAtag-pETDuet 1 | |
| 3 pRM-RBS-GFP-LVAtag-pETDuet 3 | |
| 4 pRM-RBS-GFP-LVAtag-pETDuet 4 | |
| 5 pRM-RBS-GFP-LVAtag-pETDuet 5 | |
| 6 pRM-RBS-GFP-LVAtag-pETDuet 6 | |

![](https://static.igem.org/mediawiki/2016/6/66/T--HUST-China--notebook-pRM-RBS-GFP-LVAtag-fig03.png)

## pRE-RBS-GFP-LVAtag

###### 8.23

###### Restriction Enzyme Digestion

###### Yufeng Mo

|  |  |
| --- | --- |
| Name Enzyme | |
| pRE-RBS-GFP-LVAtag XbaI and PstI | |

###### 8.23

###### DNA ligation&transformation

###### Yufeng Mo

|  |  |  |  |
| --- | --- | --- | --- |
| Name Backbone Strain Medium | | | |
| pRE-RBS-GFP-LVAtag psb1c3(XbaI and PstI cut) DH5α LB(chl added) | | | |

###### 8.24

###### Colony PCR

###### Yufeng Mo

|  |  |
| --- | --- |
| Number Name | |
| 1 pRE-RBS-GFP-LVAtag-psb1c3 | |

![](https://static.igem.org/mediawiki/2016/3/38/T--HUST-China--notebook-pRE-RBS-GFP-LVAtag-fig01.png)

###### 8.29

###### Restriction Enzyme Digestion

###### Yufeng Mo,Zhenyi Guo

|  |  |
| --- | --- |
| Name Enzyme | |
| pRE-RBS-GFP-LVAtag-psb1c3 EcoRI and PstI | |

###### 8.29

###### DNA ligation&transformation

###### Xiao Deng,Zhenyi Guo

|  |  |  |  |
| --- | --- | --- | --- |
| Name Backbone Strain Medium | | | |
| pRE-RBS-GFP-LVAtag petDuet-1(EcoRI and PstI cut) DH5α LB(Amp added) | | | |

###### 8.30

###### Colony PCR

###### Yufeng Mo

|  |  |
| --- | --- |
| Number Name | |
| 1 pRE-RBS-GFP-LVAtag-petDuet-1 1 | |
| 3 pRE-RBS-GFP-LVAtag-petDuet-1 3 | |
| 5 pRE-RBS-GFP-LVAtag-petDuet-1 5 | |
| 8 pRE-RBS-GFP-LVAtag-petDuet-1 8 | |

![](https://static.igem.org/mediawiki/2016/d/da/T--HUST-China--notebook-pRE-RBS-GFP-LVAtag-fig02.png)

## pR-RBS-GFP-LAVtag

###### 8.2

###### PCR

###### Zhenyi Guo

|  |  |
| --- | --- |
| Name Total | |
| pR-RBS-GFP-tag 50 μl | |
| pR-RBS-GFP-tag 50 μl | |
| pR-RBS-GFP-tag 50 μl | |

![](https://static.igem.org/mediawiki/2016/4/45/T--HUST-China--notebook-pR-RBS-GFP-LVAtag-fig01.png)

###### 8.2

###### Restriction Enzyme Digestion

###### Zhenyi Guo

|  |  |
| --- | --- |
| Name Enzyme | |
| psb1c3 EcoRI and PstI | |
| pR-RBS-GFP-tag EcoRI and PstI | |

![](https://static.igem.org/mediawiki/2016/b/b7/T--HUST-China--notebook-pR-RBS-GFP-LVAtag-fig02.png)

###### 8.2

###### DNA ligation&Transformation

###### Zhenyi Guo

|  |  |  |  |
| --- | --- | --- | --- |
| Name Backbone Strain Medium | | | |
| pR-RBS-GFP-tag psb1c3(EcoRI and PstI cut) DH5α LB(chl added) | | | |

###### 8.4

###### Colony PCR

###### Zhenyi Guo

|  |  |
| --- | --- |
| Number Name | |
| 2 pR-RBS-GFP-tag | |

![](https://static.igem.org/mediawiki/2016/3/3d/T--HUST-China--notebook-pR-RBS-GFP-LVAtag-fig03.png)

###### 8.24

###### Restriction Enzyme Digestion

###### Zhenyi Guo

|  |  |
| --- | --- |
| Name Enzyme | |
| pR-RBS-GFP-tag EcoRI and PstI | |

###### 8.24

###### DNA ligation&transformation

###### Yufeng Mo

|  |  |  |  |
| --- | --- | --- | --- |
| Name Backbone Strain Medium | | | |
| pR-RBS-GFP-tag pETDuet-1(EcoRI and PstI cut) DH5α LB(Amp added) | | | |

###### 8.26

###### Colony PCR

###### Yufeng Mo

|  |  |
| --- | --- |
| Number Name | |
| 4 pR-RBS-GFP-tag-pETDuet-1 4 | |
| 6 pR-RBS-GFP-tag-pETDuet-1 6 | |
| 7 pR-RBS-GFP-tag-pETDuet-1 7 | |

![](https://static.igem.org/mediawiki/2016/9/9a/T--HUST-China--notebook-pR-RBS-GFP-LVAtag-fig04.png)

## SnRK2.2

###### 9.10

###### PCR

###### Yufeng Mo

|  |  |
| --- | --- |
| Name Total | |
| SnRK2.2 1 40 μl | |
| SnRK2.2 2 40 μl | |
| SnRK2.2 0 20 μl | |

###### 9.10

###### Restriction Enzyme Digestion

###### Yufeng Mo

|  |  |
| --- | --- |
| Name Enzyme | |
| SnRK2.2 XbaI and PstI | |

###### 9.10

###### DNA ligation&transformation

###### Yufeng Mo

|  |  |  |  |
| --- | --- | --- | --- |
| Name Backbone Strain Medium | | | |
| SnRK2.2 psb1c3(XbaI and PstI cut) DH5α LB(chl added) | | | |

###### 9.12

###### Colony PCR

###### Yufeng Mo

|  |  |
| --- | --- |
| Number Name | |
| 1 SnRK2.2-psb1c3 | |

![](https://static.igem.org/mediawiki/2016/5/58/T--HUST-China--notebook-SnRK2.2-fig02.png)

## ABF2

###### 9.10

###### PCR

###### Yufeng Mo

|  |  |
| --- | --- |
| Name Total | |
| ABF2 1 40 μl | |
| ABF2 2 40 μl | |
| ABF2 0 20 μl | |

###### 9.10

###### Restriction Enzyme Digestion

###### Yufeng Mo

|  |  |
| --- | --- |
| Name Enzyme | |
| ABF2 XbaI and PstI | |

###### 9.10

###### DNA ligation&transformation

###### Yufeng Mo

|  |  |  |  |
| --- | --- | --- | --- |
| Name Backbone Strain Medium | | | |
| ABF2 psb1c3(XbaI and PstI cut) DH5α LB(chl added) | | | |

###### 9.11

###### Colony PCR

###### Yufeng Mo

|  |  |
| --- | --- |
| Number Name | |
| 2 ABF2 2 | |
| 3 ABF2 3 | |
| 4 ABF2 4 | |

![](https://static.igem.org/mediawiki/2016/8/80/T--HUST-China--notebook-ABF2-fig01.png)

## PP2CA

###### 7.25

###### PCR

###### Yufeng Mo

|  |  |
| --- | --- |
| Name Total | |
| PP2CA 1 25 μl | |
| PP2CA 2 25 μl | |
| PP2CA control group 25 μl | |

###### 7.26

###### Restriction enzyme digestion

###### Yufeng Mo

|  |  |
| --- | --- |
| Name Enzyme | |
| PP2CA XbaI and PstI | |

###### 7.26

###### DNA ligation&transformation

###### Yufeng Mo

|  |  |  |  |
| --- | --- | --- | --- |
| Name Backbone Strain Medium | | | |
| PP2CA psb1c3(Xba I and Pst I cut) E.coli DH5α LB(chl added) | | | |

###### 7.27

###### Colony PCR

###### Yufeng Mo

|  |  |
| --- | --- |
| Number Name | |
| 2 PP2CA | |

![](https://static.igem.org/mediawiki/2016/c/cd/T--HUST-China--notebook-PP2CA-fig01.png)

## prd29A

###### 9.27

###### PCR

###### Zhi Zeng

|  |  |
| --- | --- |
| Name Total | |
| prd29a 1 50 μl | |
| prd29a 2 50 μl | |
| prd29a 3 50 μl | |
| prd29a 4 50 μl | |

![](https://static.igem.org/mediawiki/2016/7/7d/T--HUST-China--notebook-prd29a-fig01.png)

###### 9.27

###### Restriction Enzyme Digestion

###### Zhenyi Guo

|  |  |
| --- | --- |
| Name Enzyme | |
| prd29a XbaI and PstI | |

![](https://static.igem.org/mediawiki/2016/0/05/T--HUST-China--notebook-prd29a-fig02.png)

###### 9.28

###### Colony PCR

###### Zhenyi Guo

|  |  |
| --- | --- |
| Number Name | |
| 1 prd29a-psb1c3 | |
| 2 prd29a-psb1c3 | |

![](https://static.igem.org/mediawiki/2016/4/4a/T--HUST-China--notebook-prd29a-fig03.png)

## CI-TT-pR-RBS-GFP-LVAtag&CII-TT-pRE-RBS-GFP-LVAtag

###### 8.17

###### Restriction Enzyme Digestion

###### Lin Di

|  |  |
| --- | --- |
| Name Enzyme | |
| Duet-pet EcoRI and PstI | |
| CI-TT-pR-RBS-GFP-LVAtag EcoRI and PstI | |
| CII-TT-pRE-RBS-GFP-LVAtag EcoRI and PstI | |

![](https://static.igem.org/mediawiki/2016/d/df/T--HUST-China--notebook-CI-TT%26CII-TT-fig01.png)![](https://static.igem.org/mediawiki/2016/b/be/T--HUST-China--notebook-CI-TT%26CII-TT-fig02.png)

###### 8.17

###### DNA ligation&Transformation

###### Lin Di

|  |  |  |  |
| --- | --- | --- | --- |
| Name Backbone Strain Medium | | | |
| CI-TTADH1-pR-RBS-GFP-LVAtag Duet-pet(EcoRI and PstI cut) DH5α LB(Amp added) | | | |
| CII-TTADH1-pRE-RBS-GFP-LVAtag Duet-pet(EcoRI and PstI cut) DH5α LB(Amp added) | | | |

###### 8.18

###### Colony PCR

###### Lin Di

|  |  |
| --- | --- |
| Number Name | |
| 6 CI-TTADH1-pR-RBS-GFP-LVAtag-petDuet-1 6 | |
| 5 CII-TTADH1-pRE-RBS-GFP-LVAtag-petDuet-1 5 | |
| 11 CII-TTADH1-pRE-RBS-GFP-LVAtag -petDuet-1 11 | |

![](https://static.igem.org/mediawiki/2016/9/91/T--HUST-China--notebook-CI-TT%26CII-TT-fig03.png)
![](https://static.igem.org/mediawiki/2016/0/02/T--HUST-China--notebook-CI-TT%26CII-TT-fig04.png)

## cro-TT-pRM-RBS-GFP-LVAtag-PSB1C3

###### 8.14

###### PCR

###### Zhi Zeng

|  |  |
| --- | --- |
| Name Total | |
| cro 80 μl | |

![](https://static.igem.org/mediawiki/2016/8/87/T--HUST-China--notebook-Cro-T-P-R-G-tag-fig01.png)

###### 8.15

###### Restriction Enzyme Digestion

###### Zhi Zeng

|  |  |
| --- | --- |
| Name Enzyme | |
| pRM-RBS-GFP-LVAtag-psb1c3 EcoRI and XbaI | |

![](https://static.igem.org/mediawiki/2016/b/b5/T--HUST-China--notebook-Cro-T-P-R-G-tag-fig02.png)

###### 8.16

###### In-Fusion

###### Zhi Zeng

|  |  |  |
| --- | --- | --- |
| Insert Vector Total | | |
| Cro&TT pRM-RBS-GFP-LVAtag-PSB1C3(Cut by EcoRⅠ&XbaⅠ) 5uL | | |

###### 8.16

###### Transformation

###### Zhi Zeng

|  |  |  |
| --- | --- | --- |
| Name Strain Medium | | |
| cro-TT-pRM-RBS-GFP-LVAtag-PSB1C3 (ligated by In-fusion) DH5α LB(Chl added) | | |

###### 8.17

###### Colony PCR

###### Zhi Zeng

|  |  |
| --- | --- |
| Name Total | |
| cro-TT-pRM-RBS-GFP-LVAtag-psb1c3 20uL | |

![](https://static.igem.org/mediawiki/2016/3/3f/T--HUST-China--notebook-Cro-T-P-R-G-tag-fig03.png)

###### 8.18

###### Restriction Enzyme Digestion

###### Zhi Zeng

|  |  |
| --- | --- |
| Name Enzyme | |
| PETDuet-1 EcoRI and PstI | |
| cro-TT-pRM-RBS-GFP-LVAtag EcoRI and PstI | |

###### 8.18

###### DNA ligation&Transformation

###### Zhi Zeng

|  |  |  |  |
| --- | --- | --- | --- |
| Name Backbone Strain Medium | | | |
| cro-TT-pRM-RBS-GFP-LVAtag pETDuet-1(EcoRI and PstI cut) DH5α LB(Amp added) | | | |

## PP2CA-TTADH1

###### 8.14

###### Restriction Enzyme Digestion

###### Ziyi Jiang

|  |  |
| --- | --- |
| Name Enzyme | |
| TTADH1-psb1c3 EcoRI and XbaI | |
| PP2CA-psb1c3 EcoRI and SpeI | |

![](https://static.igem.org/mediawiki/2016/2/2b/T--HUST-China--notebook-PP-TT-fig01.png)

###### DNA ligation&Transformation

###### Ziyi Jiang

|  |  |  |  |
| --- | --- | --- | --- |
| Name Backbone Strain Medium | | | |
| PP2CA TTADH1-psb1c3(EcoRI and XbaI cut) DH5α LB(Chl added) | | | |

###### 8.16

###### Colony PCR

###### Ziyi Jiang

|  |  |
| --- | --- |
| Number Name | |
| 5 PP2CA-TTADH1-psb1c3 5 | |
| 7 PP2CA-TTADH1-psb1c3 7 | |
| 12 PP2CA-TTADH1-psb1c3 12 | |

![](https://static.igem.org/mediawiki/2016/4/4a/T--HUST-China--notebook-PP-TT-fig02.png)

## PP2CA-TTADH1-pCyc

###### 8.22

###### Overlap PCR

###### Zhi Zeng

|  |  |
| --- | --- |
| Name Total | |
| PP2CA-TTADH1-Pcyc 80uL | |

![](https://static.igem.org/mediawiki/2016/b/b2/T--HUST-China--notebook-PP-TT-pcyc-fig01.png)

###### 8.22

###### Restriction Enzyme Digestion

###### Zhi Zeng

|  |  |
| --- | --- |
| Backbone Enzyme | |
| psb1c3 PstI and XbaI | |

![](https://static.igem.org/mediawiki/2016/5/54/T--HUST-China--notebook-PP-TT-pcyc-fig02.png)

###### 8.23

###### In-Fusion

###### Zhi Zeng

|  |  |  |
| --- | --- | --- |
| Insert Vector Total | | |
| PP2CA-TTADH1-pCyc psb1c3(Cut by PstⅠ&XbaⅠ) 5uL | | |

###### 8.23

###### Transformation

###### Zhi Zeng

|  |  |  |
| --- | --- | --- |
| Name Strain Medium | | |
| PP2CA-TTADH1-pCyc-psb1c3 (ligated by In-Fusion) DH5α LB(Chl added) | | |

###### 8.24

###### Colony PCR

###### Zhi Zeng

|  |  |
| --- | --- |
| Number Name | |
| 3 PP2CA-TTADH1-pCyc-psb1c3 | |
| 4 PP2CA-TTADH1-pCyc-psb1c3 | |
| 7 PP2CA-TTADH1-pCyc-psb1c3 | |
| 13 PP2CA-TTADH1-pCyc-psb1c3 | |
| 14 PP2CA-TTADH1-pCyc-psb1c3 | |

![](https://static.igem.org/mediawiki/2016/2/21/T--HUST-China--notebook-PP-TT-pcyc-fig03.png)

## Kozak-SnRK2.2-TTADH1-pCyc

###### 8.30

###### Overlap PCR

|  |  |
| --- | --- |
| Name Total | |
| Kozak-Snrk2.2-TTADH1-pCyc 80uL | |

![](https://static.igem.org/mediawiki/2016/5/50/T--HUST-China--notebook-K-S-T-P-fig03.png)

###### 9.5

###### In-Fusion

###### Zhi Zeng

|  |  |  |
| --- | --- | --- |
| Insert Vector Total | | |
| Kozak-Snrk2.2-TTADH1-pCyc psb1c3(Cut by PstⅠ&XbaⅠ) 5uL | | |

###### 9.5

###### Transformation

###### Zhi Zeng

|  |  |  |
| --- | --- | --- |
| Name Strain Medium | | |
| Kozak-Snrk2.2-TTADH1-pCyc-psb1c3(ligated by In-Fusion) DH5α LB(Chl added) | | |

###### 9.7

###### Colony PCR

###### Zhi Zeng

|  |  |
| --- | --- |
| Name Total | |
| Kozak-Snrk2.2-TTADH1-pCyc-psb1c3 20uL | |

![](https://static.igem.org/mediawiki/2016/5/50/T--HUST-China--notebook-K-S-T-P-fig03.png)

## placm-pRE-RBS-Cro-RBS-CII-TT

###### 9.6

###### Overlap PCR

###### Jiyue Qin

|  |  |
| --- | --- |
| Name Total | |
| Placm-pRE-RBS-Cro 50uL | |

###### 9.7

###### In-Fusion

###### Jiyue Qin

|  |  |  |
| --- | --- | --- |
| Insert Vector Total | | |
| Placm-pRE-RBS-Cro&RBS-cII-TT psb1c3(Cut by PstⅠ&XbaⅠ) 10uL | | |

###### 9.6

###### Transformation

###### Jiyue Qin

|  |  |  |
| --- | --- | --- |
| Name Strain Medium | | |
| Placm-pRE-RBS-Cro-RBS-CII-TT-psb1c3(ligated by In-Fusion) DH5α LB(Chl added) | | |

###### 9.8

###### Colony PCR

###### Jiyue Qin

|  |  |
| --- | --- |
| Number Name | |
| 1 Placm-pRE-RBS-Cro-RBS-CII-TT-psb1c3 | |

![](https://static.igem.org/mediawiki/2016/6/61/T--HUST-China--notebook-placm-p-R-Cro-fig01.png)

## patp2-RBS-CI-TTADH1-pR-RBS-CIII

###### 9.2

###### Overlap PCR

###### Jiyue Qin

|  |  |
| --- | --- |
| Name Total | |
| Patp2-RBS-CI-TT 1 50uL | |
| Patp2-RBS-CI-TT 2 50uL | |

![](https://static.igem.org/mediawiki/2016/e/e2/T--HUST-China--notebook-patp2-R-CI-TT-fig01.png)

###### 9.3

###### In-Fusion

###### Jiyue Qin

|  |  |  |
| --- | --- | --- |
| Insert Vector Total | | |
| Patp2-RBS-CI-TT&pR-RBS-CIII psb1c3(Cut by PstⅠ&XbaⅠ) 10uL | | |

###### 9.3

###### Transformation

###### Jiyue Qin

|  |  |  |
| --- | --- | --- |
| Name Strain Medium | | |
| Patp2-RBS-CI-TT-pR-RBS-CIII-psb1c3 DH5α LB(Chl added) | | |

###### 9.4

###### Colony PCR

###### Jiyue Qin

|  |  |
| --- | --- |
| Number Name | |
| 1 Patp2-RBS-CI-TT-pR-RBS-CIII-psb1c3 | |

![](https://static.igem.org/mediawiki/2016/4/4b/T--HUST-China--notebook-patp2-R-CI-TT-fig02.png)

## RBS-iLDH-TT-pRM-RBS-beta-galactosidase

###### 9.1

###### Restriction Enzyme Digestion

###### Jiyue Qin

|  |  |
| --- | --- |
| Name Enzyme | |
| GFP-LVAtag-psb1c3 XbaI and PstI | |

![](https://static.igem.org/mediawiki/2016/4/49/T--HUST-China--notebook-R-iLDH-T-p-R-β-fig01.png)

###### 9.1

###### In-Fusion

###### Jiyue Qin

|  |  |  |
| --- | --- | --- |
| Insert Vector Total | | |
| RBS-iLDH-TT&pRM-RBS-beta-galactosidase psb1c3(Cut by PstⅠ&XbaⅠ) 10uL | | |

###### 9.1

###### Transformation

###### Jiyue Qin

|  |  |  |
| --- | --- | --- |
| Name Strain Medium | | |
| RBS-iLDH-TT-pRM-RBS-beta-galactosidase-psb1c3 DH5α LB(Chl added) | | |

###### 9.2

###### Colony PCR

###### Jiyue Qin

|  |  |
| --- | --- |
| Number Name | |
| 1 RBS-iLDH-TT-pRM-RBS-beta-galactosidase-psb1c3 | |

![](https://static.igem.org/mediawiki/2016/9/96/T--HUST-China--notebook-R-iLDH-T-p-R-β-fig02.png)

## plac-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-galactosidase

###### 9.7

###### In-Fusion

###### Zhenyi Guo

|  |  |  |
| --- | --- | --- |
| Insert Vector Total | | |
| plac-pRE-RBS-Cro-RBS-CII-TT&patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-galactosidase psb1c3(Cut by PstⅠ&XbaⅠ) 10uL | | |

###### 9.7

###### Transformation

###### Zhenyi Guo

|  |  |  |
| --- | --- | --- |
| Name Strain Medium | | |
| plac-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-galactosidase DH5α LB(Chl added) | | |

###### 9.9

###### Colony PCR

###### Zhenyi Guo

|  |  |
| --- | --- |
| Number Name | |
| 1 plac-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-galactosidase | |

![](https://static.igem.org/mediawiki/2016/d/d1/T--HUST-China--notebook-Whole_circuit_of_app-fig01.png)

###### 9.11

###### Restriction Enzyme Digestion

###### Zhenyi Guo

|  |  |
| --- | --- |
| Name Enzyme | |
| pETDuet-1 EcoRI and PstI | |
| plac-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-galactosidase -psb1c3 EcoRI and PstI | |

###### 9.17

###### DNA ligation&Transformation

###### Zhenyi Guo

|  |  |  |  |
| --- | --- | --- | --- |
| Name Backbone Strain Medium | | | |
| plac-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-galactosidase pETDuet-1(cut by EcoRI and PstI) DH5α LB(Amp added) | | | |

###### 9.18

###### Colony PCR

###### Zhenyi Guo

|  |  |
| --- | --- |
| Number Name | |
| 1 plac-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-galactosidase-Duet-pet | |

![](https://static.igem.org/mediawiki/2016/c/c5/T--HUST-China--notebook-Whole_circuit_of_app-fig02.png)

## PRE-RBS-Cro-RBS-CII-TT-ptrp

###### 10.1

###### PCR

###### Zhenyi Guo

|  |  |
| --- | --- |
| Name Total | |
| PRE-RBS-Cro-RBS-CII-TT-ptrp 50 μl | |

###### 10.2

###### Restriction Enzyme Digestion

###### Zhenyi Guo

|  |  |
| --- | --- |
| Name Enzyme | |
| Psb1c3 XbalI and PstI | |

![](https://static.igem.org/mediawiki/2016/3/3d/T--HUST-China--GZY-2.jpeg)

###### 10.3

###### DNA ligation&transformation

###### Zhenyi Guo

|  |  |  |  |
| --- | --- | --- | --- |
| Name | Backbone | Strain | Medium |
| PRE-RBS-Cro-RBS-CII-TT-ptrp | psb1c3 | DH5α | LB(chl added) |

###### 10.4

###### Colony PCR

###### Zhenyi Guo

|  |  |
| --- | --- |
| Number | Name |
| 1 | PRE-RBS-Cro-RBS-CII-TT-ptrp- PSB1C3 |

![](https://static.igem.org/mediawiki/2016/6/65/T--HUST-China--GZY-4.jpeg)

## RBS-CI-TT-pR-RBS-CIII-RBS-RFPtag-TT-pRM-RBS-GFPtag

###### 10.1

###### PCR

###### Zhenyi Guo

|  |  |
| --- | --- |
| Name Total | |
| RBS-CI-TT-pR-RBS-CIII-RBS-RFPtag-TT-pRM-RBS-GFPtag 50 μl | |

###### 10.2

###### Restriction Enzyme Digestion

###### Zhenyi Guo

|  |  |
| --- | --- |
| Name Enzyme | |
| Psb1c3 | XbalI and PstI |
| RBS-CI-TT-pR-RBS-CIII-RBS-RFPtag-TT-pRM-RBS-GFPtag | XbalI and PstI |

![]( https://static.igem.org/mediawiki/2016/8/89/T--HUST-China--GZY-3.jpeg)

###### 10.3

###### DNA ligation&transformation

###### Zhenyi Guo

|  |  |  |  |
| --- | --- | --- | --- |
| Name | Backbone | Strain | Medium |
| RBS-CI-TT-pR-RBS-CIII-RBS-RFPtag-TT-pRM-RBS-GFPtag | psb1c3 | DH5α | LB(chl added) |

###### 10.4

###### Colony PCR

###### Zhenyi Guo

|  |  |
| --- | --- |
| Number | Name |
| 1 | RBS-CI-TT-pR-RBS-CIII-RBS-RFPtag-TT-pRM-RBS-GFPtag - Ppsb1c3 |

![]( https://static.igem.org/mediawiki/2016/7/7f/T--HUST-China--GZY-5.jpeg)

## pCyc-Kozak-ABF2-TTADH1-Prd29A- Kozak-GFP-LVAtag

###### 10.1

###### PCR

###### Zhenyi Guo

|  |  |
| --- | --- |
| Name Total | |
| Kozak-ABF2-TTADH1-pRD29A-Kozak-GFP-LVAtag 50 μl | |

###### 10.2

###### Restriction Enzyme Digestion

###### Zhenyi Guo

|  |  |
| --- | --- |
| Name Enzyme | |
 pCyc-psb1c3 | EcoRI and XbalI || Kozak-ABF2-TTADH1-pRD29A-Kozak-GFP-LVAtag | XbalI and PstI |

###### 10.3

###### DNA ligation&transformation

###### Zhenyi Guo

|  |  |  |  |
| --- | --- | --- | --- |
| Name | Backbone | Strain | Medium |
| Kozak-ABF2-TTADH1-pRD29A-Kozak-GFP-LVAtag | psb1c3 | DH5α | LB(chl added) |

###### 10.4

###### Colony PCR

###### Zhenyi Guo

|  |  |
| --- | --- |
| Number | Name |
| 1 | pCyc-Kozak-ABF2-TTADH1-pRD29A-Kozak-GFP-LVAtag -psb1c3 |

## RBS-ompA-iLDH-TT-pRM-RBS-beta-galactosidase-Placm-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII

###### 10.6

###### PCR

###### Zhenyi Guo

|  |  |
| --- | --- |
| Name Total | |
| RBS-ompA-iLDH 50 μl | |
| TT-pRM-RBS-beta-galactosidase | 50μl |

###### 10.7

###### Restriction Enzyme Digestion

###### Zhenyi Guo

|  |  |
| --- | --- |
| Name Enzyme | |
| Placm-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-psb1c3 EcoRI and XbalI | |

###### 10.8

###### DNA ligation&transformation

###### Zhenyi Guo

|  |  |  |  |
| --- | --- | --- | --- |
| Name | Backbone | Strain | Medium |
| RBS-ompA-iLDH、TT-pRM-RBS-beta-galactosidase | Placm-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-psb1c3 | DH5α | LB(chl added) |

###### 10.9

###### Colony PCR

###### Zhenyi Guo

|  |  |
| --- | --- |
| Number | Name |
| 1 | RBS-ompA-iLDH-TT-pRM-RBS-beta-galactosidase-Placm-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-psb1c3 |

![](https://static.igem.org/mediawiki/2016/d/d8/T--HUST-China--GZY-1.jpeg)

![](https://static.igem.org/mediawiki/2016/f/f0/T--HUST-China--returnTop.png)