iGEM 2017 — Team HUST-China

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:HUST-China/Notebook

Notebook

[![HUST_iGEM_2017_logo](https://static.igem.org/mediawiki/2017/6/64/Team_HUST_China_2017_logo.png)](https://2017.igem.org/Team%3AHUST-China)
[![HUST_iGEM_2017_logo](https://static.igem.org/mediawiki/2017/f/f6/IGEM_2017_logo.png)](https://2017.igem.org)

* [HOME](https://2017.igem.org/Team%3AHUST-China)
* PROJECT
  + [Background](https://2017.igem.org/Team%3AHUST-China/project/background)
  + [Description](https://2017.igem.org/Team%3AHUST-China/Description)
  + [Safety](https://2017.igem.org/Team%3AHUST-China/Safety)
  + [Demonstrate](https://2017.igem.org/Team%3AHUST-China/Demonstrate)
* WETLAB

  [![HUST_iGEM_2017_logo](https://static.igem.org/mediawiki/2017/6/64/Team_HUST_China_2017_logo.png)](https://2017.igem.org/Team%3AHUST-China)
  [![HUST_iGEM_2017_logo](https://static.igem.org/mediawiki/2017/f/f6/IGEM_2017_logo.png)](https://2017.igem.org)

  - [HOME](https://2017.igem.org/Team%3AHUST-China)
  - PROJECT
    * [Background](https://2017.igem.org/Team%3AHUST-China/project/background)
    * [Description](https://2017.igem.org/Team%3AHUST-China/Description)
    * [Safety](https://2017.igem.org/Team%3AHUST-China/Safety)
    * [Demonstrate](https://2017.igem.org/Team%3AHUST-China/Demonstrate)
  - WETLAB
    * [Experiments](https://2017.igem.org/Team%3AHUST-China/Experiments)
    * [Results](https://2017.igem.org/Team%3AHUST-China/Results)
    * [Interlab](https://2017.igem.org/Team%3AHUST-China/InterLab)
    * [Notebook](https://2017.igem.org/Team%3AHUST-China/Notebook)
    * [Protocols](https://2017.igem.org/Team%3AHUST-China/wetlab/protocols)
  - [MODEL](https://2017.igem.org/Team%3AHUST-China/Model)
  - PARTS
    * [Basic part](https://2017.igem.org/Team%3AHUST-China/Basic%20part)
    * [Composite part](https://2017.igem.org/Team%3AHUST-China/Composite%20part)
    * [Part collection](https://2017.igem.org/Team%3AHUST-China/Part%20collection)
  - HP
    * [Collaborations](https://2017.igem.org/Team%3AHUST-China/Collaborations)
    * [HP/Silver](https://2017.igem.org/Team%3AHUST-China/HP/Silver)
    * [Engagement](https://2017.igem.org/Team%3AHUST-China/Engagement)
  - TEAM
    * [Team Roster](https://2017.igem.org/Team%3AHUST-China/Team)
    * [Attributions](https://2017.igem.org/Team%3AHUST-China/Attributions)

# **「Notebook」**

* [Sensing part](#section1)
* [Capture part](#section2)
* [Standardization Part](#section3)

### Sensing part

#### ***PmrC-GFP*→*PmrC-GFP*-pBAD30**

**7.10**

**PCR**

**Yiyan Yu**

| Name | Total |
| --- | --- |
| PmrC-GFP | 50μl |

![demo1](https://static.igem.org/mediawiki/2017/a/a6/2017_HUST_China_notebook_img6.png "demo1")

**7.19**

**Restriction Enzyme Digestion**

**Yiyan Yu**

| Name | Enzyme |
| --- | --- |
| *PmrC-GFP* | EcoRI and Hind III |
| pBAD30 | EcoRI and Hind III |

**7.20**

**DNA ligation & transformation**

**Yiyan Yu & Qingyang Yu**

| Name | Backbone | Strain | Medium |
| --- | --- | --- | --- |
| *PmrC-GFP* | pBAD30(EcoR I and Hind III cut) | Top10 | LB(amp added) |

**7.21**

**Colony PCR**

**Yiyan Yu**

| Number | Name |
| --- | --- |
| 1 | *PmrC-GFP*-pBAD30 |
| 5 | *PmrC-GFP*-pBAD30 |
| 6 | *PmrC-GFP*-pBAD30 |

#### ***PmrA-PmrB(Fe)*→*PmrA-PmrB(Fe)*-pBAD30**

**7.8**

**PCR**

**Long Cheng**

| Name | Total |
| --- | --- |
| *PmrA-PmrB(Fe)* | 50 μl |

![demo1](https://static.igem.org/mediawiki/2017/b/b5/2017_HUST_China_notebook_img7.png "demo1")

**7.19**

**Restriction Enzyme Digestion**

**Yiyan Yu**

| Name | Enzyme |
| --- | --- |
| *PmrA-PmrB(Fe)* | EcoRI and KpnI |
| pBAD30 | EcoRI and KpnI |

**7.20**

**Restriction Enzyme Digestion**

**Yiyan Yu & Qingyang Yu**

| Name | Backbone | Strain | Medium |
| --- | --- | --- | --- |
| *PmrA-PmrB(Fe)* | pBAD30(EcoR I and Hind III cut) | Top10 | LB(amp added) |

**7.21**

**Colony PCR**

**Yiyan Yu**

| Number | Name |
| --- | --- |
| 1 | *PmrA-PmrB(Fe)*-pBAD30 |
| 5 | *PmrA-PmrB(Fe)*-pBAD30 |
| 6 | *PmrA-PmrB(Fe)*-pBAD30 |

![demo1](https://static.igem.org/mediawiki/2017/a/ac/2017_HUST_China_notebook_img8.png "demo1")

#### ***PmrA-PmrB(Fe)*-pBAD30→*PmrA-PmrB(LBTX)*-pBAD30**

**7.25**

**PCR**

**Yiyan Yu**

| Name | Total |
| --- | --- |
| *LBT* 1 to 12 | 50 μl each |

![demo1](https://static.igem.org/mediawiki/2017/e/e5/2017_HUST_China_notebook_img9.png "demo1")

**8.10**

**Point mutation**

**Xiaoyang Li**

| Name | Total |
| --- | --- |
| *PmrA-PmrB(Fe)*-pBAD30 | 50 μl |

**8.20**

**Restriction Enzyme Digestion**

**Xiaoyang Li**

| Name | Enzyme |
| --- | --- |
| *PmrA-PmrB(Fe)*-pBAD30 | AhdI and SacI |
| *LBT 1* | AhdI and SacI |
| *LBT 11* | AhdI and SacI |

![demo1](https://static.igem.org/mediawiki/2017/8/87/2017_HUST_China_notebook_img10.png "demo1")

**8.20**

**Transformation**

**Xiaoyang Li & Yating Hu**

| Name | Backbone | Strain | Medium |
| --- | --- | --- | --- |
| LBT 1 | *PmrA-PmrB(Fe)*-pBAD30(AhdI and SacI cut) | Top10 | LB(amp added) |
| LBT 11 | *PmrA-PmrB(Fe)*-pBAD30(AhdI and SacI cut) | Top10 | LB(amp added) |

**8.21**

**Colony PCR**

**Xiaoyang Li**

| Number | Name |
| --- | --- |
| 1 | *PmrA-PmrB(LBT 1)*-pBAD30 |
| 1 | *PmrA-PmrB(LBT 11)*-pBAD30 |

![demo1](https://static.igem.org/mediawiki/2017/d/d6/2017_HUST_China_notebook_img11.png "demo1")

**8.31**

**Restriction Enzyme Digestion**

**Xiaoyang Li**

| Name | Enzyme |
| --- | --- |
| *PmrA-PmrB(LBT2)* | EcoRI and KpnI |
| *PmrA-PmrB(LBT3)* | EcoRI and KpnI |
| *PmrA-PmrB(LBT4)* | EcoRI and KpnI |
| *PmrA-PmrB(LBT5)* | EcoRI and KpnI |
| *PmrA-PmrB(LBT6)* | EcoRI and KpnI |
| *PmrA-PmrB(LBT7)* | EcoRI and KpnI |
| *PmrA-PmrB(LBT8)* | EcoRI and KpnI |
| *PmrA-PmrB(LBT9)* | EcoRI and KpnI |
| *PmrA-PmrB(LBT10)* | EcoRI and KpnI |
| *PmrA-PmrB(LBT12)* | EcoRI and KpnI |

![demo1](https://static.igem.org/mediawiki/2017/6/6d/2017_HUST_China_notebook_img12.png "demo1")

**8.31**

**Transformation**

**Xiaoyang Li & Yating Hu**

| Name | Backbone | Strain | Medium |
| --- | --- | --- | --- |
| *PmrA-PmrB(LBT 2,3,4,5,6,7,8,9,10,12)* | pBAD30(EcoR I and Kpn I cut) | Top10 | LB(amp added) |

**9.3**

**Colony PCR**

**Xiaoyang Li**

| Number | Name |
| --- | --- |
| 1 | *PmrA-PmrB(LBT2)* |
| 2 | *PmrA-PmrB(LBT3)* |
| 3 | *PmrA-PmrB(LBT4)* |
| 4 | *PmrA-PmrB(LBT5)* |
| 5 | *PmrA-PmrB(LBT6)* |
| 6 | *PmrA-PmrB(LBT7)* |
| 7 | *PmrA-PmrB(LBT8)* |
| 8 | *PmrA-PmrB(LBT9)* |
| 9 | *PmrA-PmrB(LBT10)* |
| 10 | *PmrA-PmrB(LBT12)* |

![demo1](https://static.igem.org/mediawiki/2017/3/3f/2017_HUST_China_notebook_img13.png "demo1")

#### ***PmrA-PmrB(LBT X)*-pBAD30→*PmrA-PmrB(LBT X)-PmrC-GFP*-pBAD30**

**10.18**

**PCR**

**Long Cheng**

| Name | Total |
| --- | --- |
| *PmrA-PmrB(LBT 5)* | 30 μl |
| *PmrA-PmrB(LBT 9)* | 30 μl |
| *PmrA-PmrB(LBT 11)* | 30 μl |
| *PmrC-GFP* | 50 μl |

**10.19**

**SOE PCR**

**Xiaoyang Li & Qingyang Yu**

| Name | Total |
| --- | --- |
| *PmrA-PmrB(LBT 5)* & PmrC-GFP | 50 μl |
| *PmrA-PmrB(LBT 9)* & PmrC-GFP | 50 μl |
| *PmrA-PmrB(LBT 11)* & PmrC-GFP | 50 μl |

![demo1](https://static.igem.org/mediawiki/2017/2/25/2017_HUST_China_notebook_img14.png "demo1")

**10.19**

**Restriction Enzyme Digestion**

**Xiaoyang Li**

| Name | Enzyme |
| --- | --- |
| *PmrA-PmrB(LBT 5)* & *PmrC-GFP* | EcoRI and Hind III |
| *PmrA-PmrB(LBT 9)* & *PmrC-GFP* | EcoRI and Hind III |
| *PmrA-PmrB(LBT 11)* & *PmrC-GFP* | EcoRI and Hind III |
| pBAD30 | EcoRI and Hind III |

**10.19**

**DNA ligation & transformation**

**Xiaoyang Li & Yating Hu**

| Name | Backbone | Strain | Medium |
| --- | --- | --- | --- |
| *PmrA-PmrB(LBT 5)-PmrC-GFP* | pBAD30(EcoRI and Hind III cut) | Top10 | LB(amp added) |
| *PmrA-PmrB(LBT 9)-PmrC-GFP* | pBAD30(EcoRI and Hind III cut) | Top10 | LB(amp added) |
| *PmrA-PmrB(LBT 11)-PmrC-GFP* | pBAD30(EcoRI and Hind III cut) | Top10 | LB(amp added) |

**10.20**

**Colony PCR**

**Xiaoyang Li**

| Number | Name |
| --- | --- |
| 1 | *PmrA-PmrB(LBT 5)-PmrC-GFP* |
| 1 | *PmrA-PmrB(LBT 9)-PmrC-GFP* |
| 1 | *PmrA-PmrB(LBT 11)-PmrC-GFP* |

**10.19**

**SOE PCR**

**Qingyang Yu**

| Name | Total |
| --- | --- |
| *PmrA-PmrB(LBT 3)* | 50 μl |
| *PmrA-PmrB(LBT 6)* | 50 μl |
| *PmrA-PmrB(LBT 7)* | 50 μl |
| *PmrA-PmrB(LBT 10)* | 50 μl |
| *PmrA-PmrB(LBT 11)* | 50 μl |
| *PmrC-GFP* | 50 μl |

![](https://static.igem.org/mediawiki/2017/9/9d/2017_HUST_China_notebook_pic7.jpg)

**10.24**

**SOE PCR**

**Qingyang Yu**

| Name | Total |
| --- | --- |
| *PmrA-PmrB(LBT3)-PmrC-GFP* | 50 μl |
| *PmrA-PmrB(LBT6)-PmrC-GFP* | 50 μl |
| *PmrA-PmrB(LBT 7)-PmrC-GFP* | 50 μl |
| *PmrA-PmrB(LBT 9)-PmrC-GFP* | 50 μl |
| *PmrA-PmrB(LBT 10)-PmrC-GFP* | 50 μl |

![](https://static.igem.org/mediawiki/2017/a/ad/2017_HUST_China_notebook_pic8.jpg)

**10.19**

**Restriction Enzyme Digestion**

**Xiaoyang Li**

| Name | Enzyme |
| --- | --- |
| *PmrA-PmrB(LBT 3)-PmrC-GFP* | EcoRI and Hind III |
| *PmrA-PmrB(LBT 5)-PmrC-GFP* | EcoRI and Hind III |
| *PmrA-PmrB(LBT 6)-PmrC-GFP* | EcoRI and Hind III |
| *PmrA-PmrB(LBT 7)-PmrC-GFP* | EcoRI and Hind III |
| *PmrA-PmrB(LBT 9)-PmrC-GFP* | EcoRI and Hind III |
| *PmrA-PmrB(LBT 10)-PmrC-GFP* | EcoRI and Hind III |
| *PmrA-PmrB(LBT 11)-PmrC-GFP* | EcoRI and Hind III |
| pBAD30 | EcoRI and Hind III |

![](https://static.igem.org/mediawiki/2017/4/4c/2017_HUST_China_notebook_pic9.jpg)

**10.26**

**DNA ligation & transformation**

**Yiyan Yu**

| Name | Backbone | Strain | Medium |
| --- | --- | --- | --- |
| *PmrA-PmrB(LBT 3)-PmrC-GFP* | pBAD30(EcoRI and Hind III cut) | Top10 | LB(amp added) |
| *PmrA-PmrB(LBT 5)-PmrC-GFP* | pBAD30(EcoRI and Hind III cut) | Top10 | LB(amp added) |
| *PmrA-PmrB(LBT 6)-PmrC-GFP* | pBAD30(EcoRI and Hind III cut) | Top10 | LB(amp added) |
| *PmrA-PmrB(LBT 7)-PmrC-GFP* | pBAD30(EcoRI and Hind III cut) | Top10 | LB(amp added) |
| *PmrA-PmrB(LBT 9)-PmrC-GFP* | pBAD30(EcoRI and Hind III cut) | Top10 | LB(amp added) |
| *PmrA-PmrB(LBT 10)-PmrC-GFP* | pBAD30(EcoRI and Hind III cut) | Top10 | LB(amp added) |
| *PmrA-PmrB(LBT 11)-PmrC-GFP* | pBAD30(EcoRI and Hind III cut) | Top10 | LB(amp added) |

**10.28**

**Colony PCR**

**Xiaoyang Li**

| Number | Name |
| --- | --- |
| 1 | *PmrA-PmrB(LBT 5)-PmrC-GFP* |
| 2 | *PmrA-PmrB(LBT 5)-PmrC-GFP* |
| 3 | *PmrA-PmrB(LBT 5)-PmrC-GFP* |
| 4 | *PmrA-PmrB(LBT 5)-PmrC-GFP* |
| 5 | *PmrA-PmrB(LBT 5)-PmrC-GFP* |
| 6 | *PmrA-PmrB(LBT 5)-PmrC-GFP* |
| 3 | *PmrA-PmrB(LBT 6)-PmrC-GFP* |
| 4 | *PmrA-PmrB(LBT 6)-PmrC-GFP* |
| 2 | *PmrA-PmrB(LBT 7)-PmrC-GFP* |
| 3 | *PmrA-PmrB(LBT 7)-PmrC-GFP* |
| 1 | *PmrA-PmrB(LBT 9)-PmrC-GFP* |
| 2 | *PmrA-PmrB(LBT 9)-PmrC-GFP* |
| 3 | *PmrA-PmrB(LBT 9)-PmrC-GFP* |
| 4 | *PmrA-PmrB(LBT 9)-PmrC-GFP* |
| 5 | *PmrA-PmrB(LBT 9)-PmrC-GFP* |
| 6 | *PmrA-PmrB(LBT 9)-PmrC-GFP* |
| 3 | *PmrA-PmrB(LBT 10)-PmrC-GFP* |
| 4 | *PmrA-PmrB(LBT 10)-PmrC-GFP* |
| 1 | *PmrA-PmrB(LBT 11)-PmrC-GFP* |
| 2 | *PmrA-PmrB(LBT 11)-PmrC-GFP* |
| 3 | *PmrA-PmrB(LBT 11)-PmrC-GFP* |
| 4 | *PmrA-PmrB(LBT 11)-PmrC-GFP* |
| 5 | *PmrA-PmrB(LBT 11)-PmrC-GFP* |
| 6 | *PmrA-PmrB(LBT 11)-PmrC-GFP* |
| 7 | *PmrA-PmrB(LBT 11)-PmrC-GFP* |
| 8 | *PmrA-PmrB(LBT 11)-PmrC-GFP* |
| 9 | *PmrA-PmrB(LBT 11)-PmrC-GFP* |

![](https://static.igem.org/mediawiki/2017/0/07/2017_HUST_China_notebook_pic10.jpg)

### Capture part

#### ***oprF*→*oprF*-pET28a**

**7.2**

**PCR**

**Efan Wang**

| Name | Total |
| --- | --- |
| *oprF* 1 | 50 μl |
| *oprF* 2 | 50 μl |
| *oprF* 3 | 50 μl |
| *oprF* 4 | 50 μl |

![demo1](https://static.igem.org/mediawiki/2017/d/d2/2017_HUST_China_notebook_img1.png "demo1")

**7.17**

**Restriction Enzyme Digestion**

**Ziyang Xiao & Zichun Weng & Yuqi Wu**

| Name | Enzyme |
| --- | --- |
| pET28a | NheI and BamHI |
| *oprF* 1 | NheI and BamHI |
| *oprF* 2 | NheI and BamHI |

**7.27**

**DNA ligation & transformation**

**Ziyang Xiao**

| Name | Backbone | Strain | Medium |
| --- | --- | --- | --- |
| *oprF* | pET28a(BamH I and Nhe I cut) | Top10 | LB(kan added) |

**7.28**

**Colony PCR**

**Xiao Deng**

| Number | Name |
| --- | --- |
| 1 | *oprF*-pET28a |
| 2 | *oprF*-pET28a |
| 3 | *oprF*-pET28a |
| 4 | *oprF*-pET28a |
| 5 | *oprF*-pET28a |

![demo1](https://static.igem.org/mediawiki/2017/6/60/2017_HUST_China_notebook_img2.png "demo1")

#### ***3×LBT1-12* & *Sitag*→*oprF-3×LBT* X(or *Sitag*)-pET28a**

**7.22**

**PCR**

**Ziyang Xiao & Zichun Weng**

| Name | Total |
| --- | --- |
| *3 x LBT1* | 50μl |
| *3 x LBT2* | 50μl |
| *3 x LBT3* | 50μl |
| *3 x LBT4* | 50μl |
| *3 x LBT9* | 50μl |
| *3 x LBT10* | 50μl |
| *3 x LBT11* | 50μl |
| *3 x LBT12* | 50μl |

**7.27**

**PCR**

**Efan Wang & Ziyang Xiao**

| Name | Total |
| --- | --- |
| *3 x LBT5\_1* | 50μl |
| *3 x LBT5\_2* | 50μl |
| *3 x LBT6\_1* | 50μl |
| *3 x LBT6\_2* | 50μl |
| *3 x LBT7\_1* | 50μl |
| *3 x LBT7\_2* | 50μl |
| *3 x LBT8\_1* | 50μl |
| *3 x LBT8\_2* | 50μl |
| *Sitag* 1 | 50μl |
| *Sitag* 2 | 50μl |

![demo1](https://static.igem.org/mediawiki/2017/6/6c/2017_HUST_China_notebook_img3.png "demo1")

**7.27**

**Restriction Enzyme Digestion**

**Ziyang Xiao & Ting Peng**

| Name | Enzyme |
| --- | --- |
| *oprF*-pET28a | HindIII and BamHI |
| *3 x LBT2* | HindIII and BamHI |
| *3 x LBT3* | HindIII and BamHI |
| *3 x LBT4* | HindIII and BamHI |
| *3 x LBT8* | HindIII and BamHI |
| *Sitag* | HindIII and BamHI |

**8.4**

**Restriction Enzyme Digestion**

**Xiao Deng**

| Name | Enzyme |
| --- | --- |
| *3 x LBT1* | HindIII and BamHI |
| *3 x LBT5* | HindIII and BamHI |
| *3 x LBT6* | HindIII and BamHI |
| 3 x LBT7 | HindIII and BamHI |
| 3 x LBT9 | HindIII and BamHI |
| 3 x LBT10 | HindIII and BamHI |
| 3 x LBT11 | HindIII and BamHI |
| 3 x LBT12 | HindIII and BamHI |

**7.28**

**DNA ligation & transformation**

**Xiao Deng & Ting Peng**

| Name | Backbone | Stain | Medium |
| --- | --- | --- | --- |
| 3 x LBT2 | *oprF*-pET28a(BamHI and HindIII cut) | Top10 | LB(kan added) |
| 3 x LBT3 | *oprF*-pET28a(BamHI and HindIII cut) | Top10 | LB(kan added) |
| 3 x LBT4 | *oprF*-pET28a(BamHI and HindIII cut) | Top10 | LB(kan added) |
| 3 x LBT8 | *oprF*-pET28a(BamHI and HindIII cut) | Top10 | LB(kan added) |
| Sitag | *oprF*-pET28a(BamHI and HindIII cut) | Top10 | LB(kan added) |

**7.29**

**Colony PCR**

**Xiao Deng**

| Number | Name |
| --- | --- |
| 1 | *oprF*-3×LBT2-pET28a |
| 2 | *oprF*-3×LBT3-pET28a |
| 3 | *oprF*-3×LBT4-pET28a |
| 4 | *oprF*-3×LBT8-pET28a |
| 5 | *oprF*-Sitag-pET28a |

![demo1](https://static.igem.org/mediawiki/2017/3/35/2017_HUST_China_notebook_img4.png "demo1")

**8.12**

**DNA ligation & transformation**

**Xiao Deng & Nan Hu & Shaofeng Liao**

| Name | Backbone | Stain | Medium |
| --- | --- | --- | --- |
| *3 x LBT1* | *oprF*-pET28a(BamHI and HindIII cut) | Top10 | LB(kan added) |
| *3 x LBT5* | *oprF*-pET28a(BamHI and HindIII cut) | Top10 | LB(kan added) |
| *3 x LBT6* | *oprF*-pET28a(BamHI and HindIII cut) | Top10 | LB(kan added) |
| *3 x LBT7* | *oprF*-pET28a(BamHI and HindIII cut) | Top10 | LB(kan added) |
| *3 x LBT9* | *oprF*-pET28a(BamHI and HindIII cut) | Top10 | LB(kan added) |
| *3 x LBT10* | *oprF*-pET28a(BamHI and HindIII cut) | Top10 | LB(kan added) |
| *3 x LBT11* | *oprF*-pET28a(BamHI and HindIII cut) | Top10 | LB(kan added) |
| *3 x LBT12* | *oprF*-pET28a(BamHI and HindIII cut) | Top10 | LB(kan added) |

**8.13**

**Colony PCR**

**Xiao Deng**

| Number | Name |
| --- | --- |
| 1 | *oprF-3×LBT2*-pET28a |
| 2 | *oprF-3×LBT3*-pET28a |
| 3 | *oprF-3×LBT4*-pET28a |
| 4 | *oprF-3×LBT8*-pET28a |
| 5 | *oprF-Sitag*-pET28a |
| 6 | *oprF-3×LBT4*-pET28a |
| 7 | *oprF-3×LBT8*-pET28a |
| 8 | *oprF-Sitag*-pET28a |

![demo1](https://static.igem.org/mediawiki/2017/d/dc/2017_HUST_China_notebook_img5.png "demo1")

### Standardization Part

**8.20**

**PCR**

**Yubei Sun & Huiping Shi**

| Name | Total |
| --- | --- |
| *oprF-3 X LBT1* | 50 μl |
| *oprF-3 X LBT4* | 50 μl |
| *oprF-3 X LBT8* | 50 μl |
| *oprF-3 X LBT10* | 50 μl |
| *oprF-3 X LBT11* | 50 μl |
| *oprF-3 X LBT12* | 50 μl |

![demo1](https://static.igem.org/mediawiki/2017/c/c4/2017_HUST_China_notebook_img15.png "demo1")

**9.20**

**SOE PCR**

**Efan Wang**

| Name | Total |
| --- | --- |
| *oprF-3 X LBT3* | 50 μl |
| *oprF-3 X LBT6* | 50 μl |
| *oprF-3 X LBT7* | 50 μl |
| *oprF-Sitag* | 50 μl |

![demo1](https://static.igem.org/mediawiki/2017/5/5c/2017_HUST_China_notebook_img16.png "demo1")

**9.5**

**Restriction Enzyme Digestion**

**Yubei Sun & Ziyi Li**

| Name | Enzyme |
| --- | --- |
| *oprF-3 X LBT 1* | EcoRI and Spel I |
| *oprF-3 X LBT 4* | EcoRI and Spel I |
| *oprF-3 X LBT 10* | EcoRI and Spel I |
| *oprF-3 X LBT 11* | EcoRI and Spel I |
| pSB1C3 | EcoRI and Spel I |
| pSB1C3 | Xbal I and Pst I |

**9.20**

**Restriction Enzyme Digestion**

**Huiping Shi**

| Name | Enzyme |
| --- | --- |
| *oprF-3 X LBT 3* | EcoRI and Spel I |
| *oprF-3 X LBT 6* | EcoRI and Spel I |
| *oprF-3 X LBT 7* | EcoRI and Spel I |
| *oprF-3 X LBT 8* | EcoRI and Spel I |
| *oprF-3 X LBT 11* | EcoRI and Spel I |
| *oprF-3 X LBT 12* | EcoRI and Spel I |

**9.27**

**DNA ligation & transformation**

**Yubei Sun & Huiping Shi**

| Name | Backbone | Strain | Medium |
| --- | --- | --- | --- |
| *oprF-3 X LBT 1* | pSB1C3(EcoRI and Spel I cut) | DH5a | LB(chl added) |
| *oprF-3 X LBT 3* | pSB1C3(EcoRI and Spel I cut) | DH5a | LB(chl added) |
| *oprF-3 X LBT 4* | pSB1C3(EcoRI and Spel I cut) | DH5a | LB(chl added) |
| *oprF-3 X LBT 8* | pSB1C3(EcoRI and Spel I cut) | DH5a | LB(chl added) |
| *oprF-Sitag* | pSB1C3(EcoRI and Spel I cut) | DH5a | LB(chl added) |

**9.27**

**DNA ligation & transformation**

**Ziyang Xiao**

| Name | Backbone | Strain | Medium |
| --- | --- | --- | --- |
| *oprF-3 X LBT 6* | pSB1C3(EcoRI and Spel I cut) | DH5a | LB(chl added) |
| *oprF-3 X LBT 7* | pSB1C3(EcoRI and Spel I cut) | DH5a | LB(chl added) |
| *oprF-3 X LBT 10* | pSB1C3(EcoRI and Spel I cut) | DH5a | LB(chl added) |
| *oprF-3 X LBT 11* | pSB1C3(EcoRI and Spel I cut) | DH5a | LB(chl added) |
| *oprF-3 X LBT 12* | pSB1C3(EcoRI and Spel I cut) | DH5a | LB(chl added) |

**9.28**

**Colony PCR**

**Yubei Sun**

| Number | Name |
| --- | --- |
| 1 | *oprF-3×LBT1*-pSB1C3 |
| 2 | *oprF-3×LBT3*-pSB1C3 |
| 3 | *oprF-3×LBT4*-pSB1C3 |
| 4 | *oprF-3×LBT8*-pSB1C3 |
| 5 | *oprF-Sitag*-pSB1C3 |

**9.30**

**Colony PCR**

**Ziyang Xiao**

| Number | Name |
| --- | --- |
| 1 | *oprF-3×LBT6*-pSB1C3 |
| 2 | *oprF-3×LBT7*-pSB1C3 |
| 3 | *oprF-3×LBT10*-pSB1C3 |
| 4 | *oprF-3×LBT11*-pSB1C3 |
| 5 | *oprF-3×LBT12*-pSB1C3 |

#### ***PmrC-GFP*-pBAD30→*PmrC-GFP*-pSB1C3**

**10.2**

**PCR**

**Ziyang Xiao**

| Name | Total |
| --- | --- |
| *PmrC-GFP* | 30 μl |

**10.4**

**Restriction Enzyme Digestion**

**Yubei Sun**

| Name | Enzyme |
| --- | --- |
| *PmrC-GFP* | Pstl I and XbaI |

**10.5**

**DNA ligation & transformation**

**Ziyang Xiao**

| Name | Backbone | Strain | Medium |
| --- | --- | --- | --- |
| *PmrC-GFP* | pSB1C3(Pstl I and XbaI cut) | DH5a | LB(chl added) |

**10.6**

**Colony PCR**

**Ziyang Xiao**

| Number | Name |
| --- | --- |
| 1 | *PmrC-GFP*-pSB1C3 |
| 2 | *PmrC-GFP*-pSB1C3 |
| 3 | *PmrC-GFP*-pSB1C3 |
| 4 | *PmrC-GFP*-pSB1C3 |
| 5 | *PmrC-GFP*-pSB1C3 |

#### ***PmrA-PmrB(LBT X)-PmrC-GFP*-pBAD30→*PmrA-PmrB(LBT X)-PmrC-GFP*-pSB1C3**

**10.26**

**PCR**

**Ziyang Xiao**

| Name | Total |
| --- | --- |
| *PmrA-PmrB(LBT 5)PmrC-GFP* | 30 μl |
| *PmrA-PmrB(LBT 9)-PmrC-GFP* | 30 μl |
| *PmrA-PmrB(LBT 11)-PmrC-GFP* | 30 μl |

**10.4**

**Restriction Enzyme Digestion**

**Huiping Shi**

| Name | Enzyme |
| --- | --- |
| *PmrA-PmrB（LBT 5）-PmrC-GFP* | Pst I and XbaI |
| *PmrA-PmrB（LBT 9）-PmrC-GFP* | Pst I and XbaI |
| *PmrA-PmrB（LBT 11）-PmrC-GFP* | Pst I and XbaI |

**10.5**

**DNA ligation & transformation**

**Huiping Shi**

| Name | Backbone | Strain | Medium |
| --- | --- | --- | --- |
| *PmrA-PmrB（LBT 5）-PmrC-GFP* | pSB1C3(Pst I and Spel I cut) | DH5a | LB(chl added) |
| *PmrA-PmrB（LBT 9）-PmrC-GFP* | pSB1C3(Pst I and Spel I cut) | DH5a | LB(chl added) |
| *PmrA-PmrB（LBT 11）-PmrC-GFP* | pSB1C3(Pst I and Spel I cut) | DH5a | LB(chl added) |

**10.13**

**Splicing by overlap extension step 1**

**Yiyan Yu**

| Name | Total |
| --- | --- |
| *PmrA-PmrB(LBT5)* | 50μl |
| *PmrA-PmrB(LBT9)* | 50μl |
| *PmrA-PmrB(LBT11)* | 50μl |
| *PmrC-GFP* | 50μl |

![](https://static.igem.org/mediawiki/2017/a/ac/2017_HUST_China_notebook_pic1.jpg)
![](https://static.igem.org/mediawiki/2017/4/40/2017_HUST_China_notebook_pic2.jpg)

**10.14**

**Splicing by overlap extension step 2**

**Qingyang Yu**

| Name | Total |
| --- | --- |
| *PmrA-PmrB(LBT5)-PmrC-GFP* | 50μl |
| *PmrA-PmrB(LBT9)-PmrC-GFP* | 50μl |
| *PmrA-PmrB(LBT11)-PmrC-GFP* | 50μl |

![](https://static.igem.org/mediawiki/2017/d/db/2017_HUST_China_notebook_pic3.jpg)

**10.16**

**Restriction Enzyme Digestion**

**Yiyan Yu**

| Name | Enzyme |
| --- | --- |
| *PmrA-PmrB（LBT 5）-PmrC-GFP* | EcoR I & Pst I |
| *PmrA-PmrB（LBT 9）-PmrC-GFP* | EcoR I & Pst I |
| *PmrA-PmrB（LBT 11）-PmrC-GFP* | EcoR I & Pst I |

![](https://static.igem.org/mediawiki/2017/7/73/2017_HUST_China_notebook_pic4.jpg)

**10.19**

**Restriction Enzyme Digestion**

**Yiyan Yu**

| Name | Enzyme |
| --- | --- |
| *psb1c3-PmrC-GFP* | EcoR I & Pst I |

![](https://static.igem.org/mediawiki/2017/9/91/2017_HUST_China_notebook_pic5.jpg)

**10.21**

**DNA ligation & transformation**

**Ziyang Xiao**

| Name | Backbone | Strain | Medium |
| --- | --- | --- | --- |
| *PmrA-PmrB(LBT5)-PmrC-GFP* | pSB1C3(Pstl I and XbaI cut) | DH5a | LB(chl added) |
| *PmrA-PmrB(LBT9)-PmrC-GFP* | pSB1C3(Pstl I and XbaI cut) | DH5a | LB(chl added) |
| *PmrA-PmrB(LBT11)-PmrC-GFP* | pSB1C3(Pstl I and XbaI cut) | DH5a | LB(chl added) |

**10.22**

**Colony PCR**

**Kangyuan Yu**

| Number | Name |
| --- | --- |
| 4 | *PmrA-PmrB(LBT5)-PmrC-GFP* |
| 1 | *PmrA-PmrB(LBT9)-PmrC-GFP* |
| 2 | *PmrA-PmrB(LBT9)-PmrC-GFP* |
| 6 | *PmrA-PmrB(LBT9)-PmrC-GFP* |
| 2 | *PmrA-PmrB(LBT11)-PmrC-GFP* |

![](https://static.igem.org/mediawiki/2017/1/12/2017_HUST_China_notebook_pic6.jpg)

### **Acknowledgments:**

![](https://static.igem.org/mediawiki/2017/8/88/2017_HUST_China_seven_logo.png)

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)


[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:HUST-China/Experiments

Experiment

[![HUST_iGEM_2017_logo](https://static.igem.org/mediawiki/2017/6/64/Team_HUST_China_2017_logo.png)](https://2017.igem.org/Team%3AHUST-China)
[![HUST_iGEM_2017_logo](https://static.igem.org/mediawiki/2017/f/f6/IGEM_2017_logo.png)](https://2017.igem.org)

* [HOME](https://2017.igem.org/Team%3AHUST-China)
* PROJECT
  + [Background](https://2017.igem.org/Team%3AHUST-China/project/background)
  + [Description](https://2017.igem.org/Team%3AHUST-China/Description)
  + [Safety](https://2017.igem.org/Team%3AHUST-China/Safety)
  + [Demonstrate](https://2017.igem.org/Team%3AHUST-China/Demonstrate)
* WETLAB

  [![HUST_iGEM_2017_logo](https://static.igem.org/mediawiki/2017/6/64/Team_HUST_China_2017_logo.png)](https://2017.igem.org/Team%3AHUST-China)
  [![HUST_iGEM_2017_logo](https://static.igem.org/mediawiki/2017/f/f6/IGEM_2017_logo.png)](https://2017.igem.org)

  - [HOME](https://2017.igem.org/Team%3AHUST-China)
  - PROJECT
    * [Background](https://2017.igem.org/Team%3AHUST-China/project/background)
    * [Description](https://2017.igem.org/Team%3AHUST-China/Description)
    * [Safety](https://2017.igem.org/Team%3AHUST-China/Safety)
    * [Demonstrate](https://2017.igem.org/Team%3AHUST-China/Demonstrate)
  - WETLAB
    * [Experiments](https://2017.igem.org/Team%3AHUST-China/Experiments)
    * [Results](https://2017.igem.org/Team%3AHUST-China/Results)
    * [Interlab](https://2017.igem.org/Team%3AHUST-China/InterLab)
    * [Notebook](https://2017.igem.org/Team%3AHUST-China/Notebook)
    * [Protocols](https://2017.igem.org/Team%3AHUST-China/wetlab/protocols)
  - [MODEL](https://2017.igem.org/Team%3AHUST-China/Model)
  - PARTS
    * [Basic part](https://2017.igem.org/Team%3AHUST-China/Basic%20part)
    * [Composite part](https://2017.igem.org/Team%3AHUST-China/Composite%20part)
    * [Part collection](https://2017.igem.org/Team%3AHUST-China/Part%20collection)
  - HP
    * [Collaborations](https://2017.igem.org/Team%3AHUST-China/Collaborations)
    * [HP/Silver](https://2017.igem.org/Team%3AHUST-China/HP/Silver)
    * [Engagement](https://2017.igem.org/Team%3AHUST-China/Engagement)
  - TEAM
    * [Team Roster](https://2017.igem.org/Team%3AHUST-China/Team)
    * [Attributions](https://2017.igem.org/Team%3AHUST-China/Attributions)

# **「Experiment」**

* [Sensing part](#section1)
* [Enrichment part
   and recycling part](#section2)
* [The whole circuit](#section3)
* [Detection of
  sensing part](#section4)
* [Detection of adsorption
  capacity of LBTs](#section5)

### Sensing part

The sensing part is to achieve the awareness of the engineering bacteria on the rare earth ions in the environment.

#### **①Amplify**

*******PmrA-PmrB(Fe)*******、  ****PmrC-GFP****  gene through the pcr protocol.

#### **②Plasmid Construction**

Coding sequences  *PmrA-PmrB(Fe)* and  ****PmrC-GFP**** were cloned into the expression vector pBAD30 by restriction enzyme digestion and DNA ligation.

![experiment_circuit](https://static.igem.org/mediawiki/2017/d/d5/2017_HUST_China_Experiment_image1.png)

Then we transformed these vectors into Ecoli BL21 cells.

#### **③Detection**

Arabinose was used to induce expression of PmrA-PmrB-PmrC system.

We used the original  **PmrA-PmrB(Fe)** - ****PmrC-GFP**** circuit to test whether our
PmrA-PmrB-PmrC system works in our plasmid, then the expression and efficacy
of *LBT1-12* were tested .

| gene circuit | vector | description |
| --- | --- | --- |
| *PmrA-PmrB(Fe)-PmrC-GFP* | pBAD30 | Test whether our PmrABC system works in our plasmid |
| *PmrA-PmrB(LBT1)-PmrC-GFP* | pBAD30 | Detecting effectiveness of protein LBT1 |
| *PmrA-PmrB(LBT2)-PmrC-GFP* | pBAD30 | Detecting effectiveness of protein LBT2 |
| *PmrA-PmrB(LBT3)-PmrC-GFP* | pBAD30 | Detecting effectiveness of protein LBT3 |
| *PmrA-PmrB(LBT4)-PmrC-GFP* | pBAD30 | Detecting effectiveness of protein LBT4 |
| *PmrA-PmrB(LBT5)-PmrC-GFP* | pBAD30 | Detecting effectiveness of protein LBT5 |
| *PmrA-PmrB(LBT6)-PmrC-GFP* | pBAD30 | Detecting effectiveness of protein LBT6 |
| *PmrA-PmrB(LBT7)-PmrC-GFP* | pBAD30 | Detecting effectiveness of protein LBT7 |
| *PmrA-PmrB(LBT8)-PmrC-GFP* | pBAD30 | Detecting effectiveness of protein LBT8 |
| *PmrA-PmrB(LBT9)-PmrC-GFP* | pBAD30 | Detecting effectiveness of protein LBT9 |
| *PmrA-PmrB(LBT10)-PmrC-GFP* | pBAD30 | Detecting effectiveness of protein LBT10 |
| *PmrA-PmrB(LBT11)-PmrC-GFP* | pBAD30 | Detecting effectiveness of protein LBT11 |
| *PmrA-PmrB(LBT12)-PmrC-GFP* | pBAD30 | Detecting effectiveness of protein LBT12 |

### Enrichment part and recycling part

**The Capture part is to capture the rare earth ions in the environment and to anchor our cells over the silicon net.**

#### **①With appropriate primers, PCR was carried out to amplify target gene *oprF-LBT1-12* and *oprF-Sitag*.**

#### **②Then we constructed two vectors to achieve expression of *LBT* and *Sitag*.**

![demo1](https://static.igem.org/mediawiki/2017/7/73/2017_HUST_China_Experiment_image2.png "demo1")
![demo1](https://static.igem.org/mediawiki/2017/7/78/2017_HUST_China_Experiment_image3.png "demo1")

We cloned the sequence of *oprF-LBT* and *Sitag* into the vector pET28α in order to make the sequence be tagged N-terminally with a 6×His tag. 6×His tag is an affinity tag which allows the tagged recombinant protein to be purified in the process of affinity purification.

#### **③Detection**

In the meanwhile, we added the FLAG tag to test whether the *LBTs* and the *Sitag* was expressed on the cell membrane.

### Construction of the whole circuit

*PmrA-PmrB(Fe)-PmrC-GFP* All of our part should be built in one plasmid. Therefore, we use endonuclease and ligase to replace the *GFP* by *oprF-LBT* and *oprF-Sitag*.

![demo1](https://static.igem.org/mediawiki/2017/5/58/2017_HUST_China_Experiment_image4.png "demo1")

### Detection of sensing part

We use our medium containing a small amount of rare earth ions to culture our E. coli. When our engineered E. coli sense the existence of rare earth ions, the PmrC promoter will activate the transcription of the *GFP* gene. Through the detection of *GFP* expression, we can know the efficacy of the sensing part.

### Detection of adsorption capacity of LBTs

A method of precipitation titration is used to detect the level of rare earth ions adsorption. We add a certain concentration of bacteria to the solution containing different concentrations of rare earth ions. Then centrifuge to remove the bacteria in the solution.

![demo1](https://static.igem.org/mediawiki/2017/1/1a/2017_HUST_China_Experiment_image5.png "demo1")

In order to make the terbium ions sufficiently precipitated, we add excess base. Rinse the precipitate and dissolve the precipitate with 0.1 M hydrochloric acid titration. In this way, we can know the amount of terbium ions which have already adsorbed on the surface of the bacteria.

### **Acknowledgments:**

![](https://static.igem.org/mediawiki/2017/8/88/2017_HUST_China_seven_logo.png)

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:HUST-China/Results

Results

[![HUST_iGEM_2017_logo](https://static.igem.org/mediawiki/2017/6/64/Team_HUST_China_2017_logo.png)](https://2017.igem.org/Team%3AHUST-China)
[![HUST_iGEM_2017_logo](https://static.igem.org/mediawiki/2017/f/f6/IGEM_2017_logo.png)](https://2017.igem.org)

* [HOME](https://2017.igem.org/Team%3AHUST-China)
* PROJECT
  + [Background](https://2017.igem.org/Team%3AHUST-China/project/background)
  + [Description](https://2017.igem.org/Team%3AHUST-China/Description)
  + [Safety](https://2017.igem.org/Team%3AHUST-China/Safety)
  + [Demonstrate](https://2017.igem.org/Team%3AHUST-China/Demonstrate)
* WETLAB

  [![HUST_iGEM_2017_logo](https://static.igem.org/mediawiki/2017/6/64/Team_HUST_China_2017_logo.png)](https://2017.igem.org/Team%3AHUST-China)
  [![HUST_iGEM_2017_logo](https://static.igem.org/mediawiki/2017/f/f6/IGEM_2017_logo.png)](https://2017.igem.org)

  - [HOME](https://2017.igem.org/Team%3AHUST-China)
  - PROJECT
    * [Background](https://2017.igem.org/Team%3AHUST-China/project/background)
    * [Description](https://2017.igem.org/Team%3AHUST-China/Description)
    * [Safety](https://2017.igem.org/Team%3AHUST-China/Safety)
    * [Demonstrate](https://2017.igem.org/Team%3AHUST-China/Demonstrate)
  - WETLAB
    * [Experiments](https://2017.igem.org/Team%3AHUST-China/Experiments)
    * [Results](https://2017.igem.org/Team%3AHUST-China/Results)
    * [Interlab](https://2017.igem.org/Team%3AHUST-China/InterLab)
    * [Notebook](https://2017.igem.org/Team%3AHUST-China/Notebook)
    * [Protocols](https://2017.igem.org/Team%3AHUST-China/wetlab/protocols)
  - [MODEL](https://2017.igem.org/Team%3AHUST-China/Model)
  - PARTS
    * [Basic part](https://2017.igem.org/Team%3AHUST-China/Basic%20part)
    * [Composite part](https://2017.igem.org/Team%3AHUST-China/Composite%20part)
    * [Part collection](https://2017.igem.org/Team%3AHUST-China/Part%20collection)
  - HP
    * [Collaborations](https://2017.igem.org/Team%3AHUST-China/Collaborations)
    * [HP/Silver](https://2017.igem.org/Team%3AHUST-China/HP/Silver)
    * [Engagement](https://2017.igem.org/Team%3AHUST-China/Engagement)
  - TEAM
    * [Team Roster](https://2017.igem.org/Team%3AHUST-China/Team)
    * [Attributions](https://2017.igem.org/Team%3AHUST-China/Attributions)

# **「Results」**

### Capture part

#### **1.1 SDS-PAGE verification for *oprF-3\*LBT 4* & *oprF-Sitag* expression**

Whether the oprF-3\*LBT or oprF-Sitag can be expressed or not needs to be detected by SDS-PAGE.

![](https://static.igem.org/mediawiki/2017/b/b7/2017_HUST_China_demostrate002.png "demo1")
![](https://static.igem.org/mediawiki/2017/c/ce/2017_HUST_China_demostrate003.png "demo1")

Firstly, we need to identify that *OprF-3\* LBT4* and *OprF-Sitag* are expressed on the surface of the E. coli. We centrifugated the bacteria solution and got the whole test BL21 cells induced by IPTG. As control, we also extracted the not induced BL21 cells. Then we separate the protein of the whole cell by SDS-PAGE.

Figure 1 shows an obvious ~33kDa protein bands in test lane, which cannot be found in the control lane. This result proves that the test BL21 can produce OprF-3\* LBT 4.

Figure 2 shows an obvious ~57kDa protein bands in test lane, which cannot be found in the not induced lane. This result proves that the test BL21 can produce OprF-Sitag.

#### **1.2 Verification of Cell Surface Display System by Immunofluorescent assay**

Then we did immunofluorescence assay to figure out whether the cell surface display system works or not.

![](https://static.igem.org/mediawiki/2017/c/cc/2017_HUST_China_demostrate004.png "demo1")

Figure 3 shows the result of our verification of our cell surface display system. Some cells can show a considerable fluorescent intensity, while some perform partial or weak fluorescence which can not be detected by our microscope camera. But this result still successfully demonstrated the cell surface display of our LBTs.

#### **1.3 Verification of Tb3+ capturing by precipitation titration of remained Tb3+**

To measure REEBOT’s ability to capture Tb ions, one kind of lanthanide ions, we have done a precipitation titration of Tb hydroxide. We first add REEBOT into Tb ion solution. Each of them has certain concentration and volume. After incubation, we discard the bacteria, and then titrate the solution with sodium hydroxide. The less precipitation of Tb hydroxide generates, the more Tb ions are captured by the bacteria.

![](https://static.igem.org/mediawiki/2017/b/b1/2017_HUST_China_demostrate005.png "demo1")
![](https://static.igem.org/mediawiki/2017/6/64/2017_HUST_China_demostrate006.png "demo1")

Figure 4 shows that it is significant difference in the amount of precipitation. The amount of precipitation in WT group(with distilled water added) is much less than that in the test group.

To get some more detailed and convincing results, we did a precipitation-dissolving titration with 0.1M hydrochloric acid. Figure 5 can show a result that the test group(with our engineered bacteria) is much different from the control group. After our engineered bacteria work a while in sample, the terbium ions concentration is significantly reduced.

### **Acknowledgments:**

![](https://static.igem.org/mediawiki/2017/8/88/2017_HUST_China_seven_logo.png)

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)