iGEM 2019 — Team Fudan-TSI

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# Team:Fudan-TSI/Notebook

Notebook | 2019 iGEM Team:Fudan-TSI

[![2019 team logo](https://static.igem.org/mediawiki/2019/d/d3/T--Fudan-TSI--HomepageLogo.gif)](/Team%3AFudan-TSI)

* Project
* Results
* Model
* Parts
* Human practices
* Team
* [Judging](/Team%3AFudan-TSI/Judging)

* [Background](/Team%3AFudan-TSI/Description)
* [Design](/Team%3AFudan-TSI/Design)
* [Experiments](/Team%3AFudan-TSI/Experiments)
* [Applied design](/Team%3AFudan-TSI/Applied_Design)

* [Reverse transcription](/Team%3AFudan-TSI/Demonstrate#ReverseTranscription)
* [Recombination](/Team%3AFudan-TSI/Demonstrate#Recombination)
* [Demonstration](/Team%3AFudan-TSI/Demonstrate)
* [Measurement](/Team%3AFudan-TSI/Measurement)
* [Notebook](/Team%3AFudan-TSI/Notebook)

* [Modeling](/Team%3AFudan-TSI/Model)
* [Software](/Team%3AFudan-TSI/Software)
* [Hardware](/Team%3AFudan-TSI/Hardware)

* [Basic parts](/Team%3AFudan-TSI/Basic_Part)
* [Composite parts](/Team%3AFudan-TSI/Composite_Part)
* [Part improvement](/Team%3AFudan-TSI/Improve)
* [Part collection](/Team%3AFudan-TSI/Part_Collection)

* [Public engagement](/Team%3AFudan-TSI/Public_Engagement)
* [Integrated HP](/Team%3AFudan-TSI/Human_Practices#IntegratedHumanPractice)
* [Collaborations](/Team%3AFudan-TSI/Collaborations)
* [Safety](/Team%3AFudan-TSI/Safety)

* [Members](/Team%3AFudan-TSI/Team)
* [Attributions](/Team%3AFudan-TSI/Attributions)
* [Acknowledge](/Team%3AFudan-TSI/Team#Acknowledge)
* [Heritage](/Team%3AFudan-TSI/Heritage)

* Notebook
* + On this page
  + [section1](#section1)
  + [section2](#section2)
  + [section3](#section3)
  + [section4](#section4)
  + Team: Fudan-TSI
  + Project

    - [Background](/Team%3AFudan-TSI/Description)
    - [Design](/Team%3AFudan-TSI/Design)
    - [Experiments](/Team%3AFudan-TSI/Experiments)
    - [Applied design](/Team%3AFudan-TSI/Applied_Design)
    - [Judging](/Team%3AFudan-TSI/Judging)
  + Results

    - [Reverse transcription](/Team%3AFudan-TSI/Demonstrate#ReverseTranscription)
    - [Recombination](/Team%3AFudan-TSI/Demonstrate#Recombination)
    - [Demonstration](/Team%3AFudan-TSI/Demonstrate)
    - [Measurement](/Team%3AFudan-TSI/Measurement)
    - [Notebook](/Team%3AFudan-TSI/Notebook)
  + Model

    - [Modeling](/Team%3AFudan-TSI/Model)
    - [Software](/Team%3AFudan-TSI/Software)
    - [Hardware](/Team%3AFudan-TSI/Hardware)
  + Parts

    - [Basic parts](/Team%3AFudan-TSI/Basic_Part)
    - [Composite parts](/Team%3AFudan-TSI/Composite_Part)
    - [Part improvement](/Team%3AFudan-TSI/Improve)
    - [Part collection](/Team%3AFudan-TSI/Part_Collection)
  + Human practices

    - [Public engagement](/Team%3AFudan-TSI/Public_Engagement)
    - [Integrated HP](/Team%3AFudan-TSI/Human_Practices#IntegratedHumanPractice)
    - [Collaborations](/Team%3AFudan-TSI/Collaborations)
    - [Safety](/Team%3AFudan-TSI/Safety)
  + Team

    - [Members](/Team%3AFudan-TSI/Team)
    - [Attributions](/Team%3AFudan-TSI/Attributions)
    - [Heritage](/Team%3AFudan-TSI/Heritage)

# Notebook

We hereby present a toolbox for *in vivo* continuous mutation library construction. Our toolbox is orthogonal and provides a wide range of applications among various species.

![cover notebook](https://static.igem.org/mediawiki/2019/4/4b/T--Fudan-TSI--coverNotebook.gif)

Here is [the link](https://static.igem.org/mediawiki/2019/a/a3/T--Fudan-TSI--Notebook.pdf) to download the file above.

[![2019 Team:Fudan-TSI logo white](https://static.igem.org/mediawiki/2019/0/0f/T--Fudan-TSI--LogoGrey.gif)](https://2019.igem.org/Team%3AFudan-TSI) [![Fudan University](https://static.igem.org/mediawiki/2018/f/f7/T--Fudan--schoolLogo.png)](http://www.fudan.edu.cn/en/) [![School of Life Sciences, Fudan University](https://static.igem.org/mediawiki/2018/1/1d/T--Fudan--schoolOfLifeSciencesIcon.png)](http://life.fudan.edu.cn/) [![Yunfeng Capital](https://static.igem.org/mediawiki/2018/e/e2/T--Fudan--yunfengLogo.png)](http://www.yfc.cn/en/)

### R-Evolution: an *in vivo* sequence-specific toolbox for continuous mutagenesis

[Project](/Team%3AFudan-TSI/Description)

* [Background](/Team%3AFudan-TSI/Description)
* [Design](/Team%3AFudan-TSI/Design)
* [Experiments](/Team%3AFudan-TSI/Experiments)
* [Applied design](/Team%3AFudan-TSI/Applied_Design)
* [Judging](/Team%3AFudan-TSI/Judging)

[Results](/Team%3AFudan-TSI/Demonstrate)

* [Reverse transcription](/Team%3AFudan-TSI/Demonstrate#ReverseTranscription)
* [Recombination](/Team%3AFudan-TSI/Demonstrate#Recombination)
* [Demonstration](/Team%3AFudan-TSI/Demonstrate)
* [Measurement](/Team%3AFudan-TSI/Measurement)
* [Notebook](/Team%3AFudan-TSI/Notebook)

[Model](/Team%3AFudan-TSI/Model)

* [Modeling](/Team%3AFudan-TSI/Model)
* [Software](/Team%3AFudan-TSI/Software)
* [Hardware](/Team%3AFudan-TSI/Hardware)

[Parts](/Team%3AFudan-TSI/Parts)

* [Basic parts](/Team%3AFudan-TSI/Basic_Part)
* [Composite parts](/Team%3AFudan-TSI/Composite_Part)
* [Part improvement](/Team%3AFudan-TSI/Improve)
* [Part collection](/Team%3AFudan-TSI/Part_Collection)

[Outreach](/Team%3AFudan-TSI/Human_Practices)

* [Public engagement](/Team%3AFudan-TSI/Public_Engagement)
* [Integrated HP](/Team%3AFudan-TSI/Human_Practices#IntegratedHumanPractice)
* [Collaborations](/Team%3AFudan-TSI/Collaborations)
* [Safety](/Team%3AFudan-TSI/Safety)

[Team](/Team%3AFudan-TSI/Team)

* [Members](/Team%3AFudan-TSI/Team)
* [Attributions](/Team%3AFudan-TSI/Attributions)
* [Acknowledge](/Team%3AFudan-TSI/Team#Acknowledge)
* [Heritage](/Team%3AFudan-TSI/Heritage)

Life Sci Bldg, 2005 Songhu Rd, Shanghai

+86-21-31246727

igem@fudan.edu.cn

Fudan\_iGEM


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

# Team:Fudan-TSI/Experiments

Experiments | 2019 iGEM Team:Fudan-TSI

[![2019 team logo](https://static.igem.org/mediawiki/2019/d/d3/T--Fudan-TSI--HomepageLogo.gif)](/Team%3AFudan-TSI)

* Project
* Results
* Model
* Parts
* Human practices
* Team
* [Judging](/Team%3AFudan-TSI/Judging)

* [Background](/Team%3AFudan-TSI/Description)
* [Design](/Team%3AFudan-TSI/Design)
* [Experiments](/Team%3AFudan-TSI/Experiments)
* [Applied design](/Team%3AFudan-TSI/Applied_Design)

* [Reverse transcription](/Team%3AFudan-TSI/Demonstrate#ReverseTranscription)
* [Recombination](/Team%3AFudan-TSI/Demonstrate#Recombination)
* [Demonstration](/Team%3AFudan-TSI/Demonstrate)
* [Measurement](/Team%3AFudan-TSI/Measurement)
* [Notebook](/Team%3AFudan-TSI/Notebook)

* [Modeling](/Team%3AFudan-TSI/Model)
* [Software](/Team%3AFudan-TSI/Software)
* [Hardware](/Team%3AFudan-TSI/Hardware)

* [Basic parts](/Team%3AFudan-TSI/Basic_Part)
* [Composite parts](/Team%3AFudan-TSI/Composite_Part)
* [Part improvement](/Team%3AFudan-TSI/Improve)
* [Part collection](/Team%3AFudan-TSI/Part_Collection)

* [Public engagement](/Team%3AFudan-TSI/Public_Engagement)
* [Integrated HP](/Team%3AFudan-TSI/Human_Practices#IntegratedHumanPractice)
* [Collaborations](/Team%3AFudan-TSI/Collaborations)
* [Safety](/Team%3AFudan-TSI/Safety)

* [Members](/Team%3AFudan-TSI/Team)
* [Attributions](/Team%3AFudan-TSI/Attributions)
* [Acknowledge](/Team%3AFudan-TSI/Team#Acknowledge)
* [Heritage](/Team%3AFudan-TSI/Heritage)

* Experiments
* + Team: Fudan-TSI
  + Project

    - [Background](/Team%3AFudan-TSI/Description)
    - [Design](/Team%3AFudan-TSI/Design)
    - [Experiments](/Team%3AFudan-TSI/Experiments)
    - [Applied design](/Team%3AFudan-TSI/Applied_Design)
    - [Judging](/Team%3AFudan-TSI/Judging)
  + Results

    - [Reverse transcription](/Team%3AFudan-TSI/Demonstrate#ReverseTranscription)
    - [Recombination](/Team%3AFudan-TSI/Demonstrate#Recombination)
    - [Demonstration](/Team%3AFudan-TSI/Demonstrate)
    - [Measurement](/Team%3AFudan-TSI/Measurement)
    - [Notebook](/Team%3AFudan-TSI/Notebook)
  + Model

    - [Modeling](/Team%3AFudan-TSI/Model)
    - [Software](/Team%3AFudan-TSI/Software)
    - [Hardware](/Team%3AFudan-TSI/Hardware)
  + Parts

    - [Basic parts](/Team%3AFudan-TSI/Basic_Part)
    - [Composite parts](/Team%3AFudan-TSI/Composite_Part)
    - [Part improvement](/Team%3AFudan-TSI/Improve)
    - [Part collection](/Team%3AFudan-TSI/Part_Collection)
  + Human practices

    - [Public engagement](/Team%3AFudan-TSI/Public_Engagement)
    - [Integrated HP](/Team%3AFudan-TSI/Human_Practices#IntegratedHumanPractice)
    - [Collaborations](/Team%3AFudan-TSI/Collaborations)
    - [Safety](/Team%3AFudan-TSI/Safety)
  + Team

    - [Members](/Team%3AFudan-TSI/Team)
    - [Attributions](/Team%3AFudan-TSI/Attributions)
    - [Heritage](/Team%3AFudan-TSI/Heritage)

# Experiments

We showed that our toolbox is orthogonal and provided a wide range of applications among various species. R-Evolution could mutate coding sequences and regulatory sequences, which enables the evolution of individual proteins or multiple targets at a time.

![cover not completely uploaded @@@@](https://static.igem.org/mediawiki/2019/f/f3/T--Fudan-TSI--coverExperiment.gif)

2019 Team Fudan-TSI works at the laboratory of Prof. Liang Cai in the School of Life Sciences of Fudan University. We mainly use the experimental devices and protocols within the laboratory, which are performed and optimized through years of practice.

Particularly, the fluorescence intensity and OD600 measurement and calibration protocols we use are from iGEM official measurement protocols.

Here is [the link](https://static.igem.org/mediawiki/2019/6/65/T--Fudan-TSI--Protocols.pdf) to download the file above.

[![2019 Team:Fudan-TSI logo white](https://static.igem.org/mediawiki/2019/0/0f/T--Fudan-TSI--LogoGrey.gif)](https://2019.igem.org/Team%3AFudan-TSI) [![Fudan University](https://static.igem.org/mediawiki/2018/f/f7/T--Fudan--schoolLogo.png)](http://www.fudan.edu.cn/en/) [![School of Life Sciences, Fudan University](https://static.igem.org/mediawiki/2018/1/1d/T--Fudan--schoolOfLifeSciencesIcon.png)](http://life.fudan.edu.cn/) [![Yunfeng Capital](https://static.igem.org/mediawiki/2018/e/e2/T--Fudan--yunfengLogo.png)](http://www.yfc.cn/en/)

### R-Evolution: an *in vivo* sequence-specific toolbox for continuous mutagenesis

[Project](/Team%3AFudan-TSI/Description)

* [Background](/Team%3AFudan-TSI/Description)
* [Design](/Team%3AFudan-TSI/Design)
* [Experiments](/Team%3AFudan-TSI/Experiments)
* [Applied design](/Team%3AFudan-TSI/Applied_Design)
* [Judging](/Team%3AFudan-TSI/Judging)

[Results](/Team%3AFudan-TSI/Demonstrate)

* [Reverse transcription](/Team%3AFudan-TSI/Demonstrate#ReverseTranscription)
* [Recombination](/Team%3AFudan-TSI/Demonstrate#Recombination)
* [Demonstration](/Team%3AFudan-TSI/Demonstrate)
* [Measurement](/Team%3AFudan-TSI/Measurement)
* [Notebook](/Team%3AFudan-TSI/Notebook)

[Model](/Team%3AFudan-TSI/Model)

* [Modeling](/Team%3AFudan-TSI/Model)
* [Software](/Team%3AFudan-TSI/Software)
* [Hardware](/Team%3AFudan-TSI/Hardware)

[Parts](/Team%3AFudan-TSI/Parts)

* [Basic parts](/Team%3AFudan-TSI/Basic_Part)
* [Composite parts](/Team%3AFudan-TSI/Composite_Part)
* [Part improvement](/Team%3AFudan-TSI/Improve)
* [Part collection](/Team%3AFudan-TSI/Part_Collection)

[Outreach](/Team%3AFudan-TSI/Human_Practices)

* [Public engagement](/Team%3AFudan-TSI/Public_Engagement)
* [Integrated HP](/Team%3AFudan-TSI/Human_Practices#IntegratedHumanPractice)
* [Collaborations](/Team%3AFudan-TSI/Collaborations)
* [Safety](/Team%3AFudan-TSI/Safety)

[Team](/Team%3AFudan-TSI/Team)

* [Members](/Team%3AFudan-TSI/Team)
* [Attributions](/Team%3AFudan-TSI/Attributions)
* [Acknowledge](/Team%3AFudan-TSI/Team#Acknowledge)
* [Heritage](/Team%3AFudan-TSI/Heritage)

Life Sci Bldg, 2005 Songhu Rd, Shanghai

+86-21-31246727

igem@fudan.edu.cn

Fudan\_iGEM

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

# Team:Fudan-TSI/Results

![](https://static.igem.org/mediawiki/2019/d/d3/T--Fudan-TSI--HomepageLogo.gif)

![](https://static.igem.org/mediawiki/2019/thumb/a/ae/T--Fudan-TSI--mobileCtrl.gif/683px-T--Fudan-TSI--mobileCtrl.gif)

* ![](https://static.igem.org/mediawiki/2019/d/d3/T--Fudan-TSI--HomepageLogo.gif)

+ [Home](https://2019.igem.org/Team%3AFudan-TSI)
+ [Project](https://2019.igem.org/Team%3AFudan-TSI/Description)

  - [Description](https://2019.igem.org/Team%3AFudan-TSI/Description)
  - [Design](https://2019.igem.org/Team%3AFudan-TSI/Design)
  - [Applied Design](https://2019.igem.org/Team%3AFudan-TSI/Applied_Design)
  - [Experiment](https://2019.igem.org/Team%3AFudan-TSI/Experiments)
+ [Results](https://2019.igem.org/Team%3AFudan-TSI/Results)

  - [Reverse Transcription](https://2019.igem.org/Team%3AFudan-TSI/Results/Reverse_Transcription)
  - [Recombination](https://2019.igem.org/Team%3AFudan-TSI/Results/Recombination)
  - [Demonstration](https://2019.igem.org/Team%3AFudan-TSI/Demonstrate)
  - [Measurement](https://2019.igem.org/Team%3AFudan-TSI/Measurement)
  - [Notebook](https://2019.igem.org/Team%3AFudan-TSI/Notebook)
+ [Model](https://2019.igem.org/Team%3AFudan-TSI/Model)

  - [Modeling](https://2019.igem.org/Team%3AFudan-TSI/Model)
  - [Software](https://2019.igem.org/Team%3AFudan-TSI/Software)
  - [Hardware](https://2019.igem.org/Team%3AFudan-TSI/Hardware)
+ [Parts](https://2019.igem.org/Team%3AFudan-TSI/Parts)

  - [Basic Parts](https://2019.igem.org/Team%3AFudan-TSI/Basic_Part)
  - [Composite Parts](https://2019.igem.org/Team%3AFudan-TSI/Composite_Part)
  - [Improved Parts](https://2019.igem.org/Team%3AFudan-TSI/Improve)
  - [Part Collection](https://2019.igem.org/Team%3AFudan-TSI/Part_Collection)
+ [Human Practices](https://2019.igem.org/Team%3AFudan-TSI/Human_Practices)

  - [Education &
    Public Engagement](https://2019.igem.org/Team%3AFudan-TSI/Public_Engagement)
  - [Integrated
    Human Practice](https://2019.igem.org/Team%3AFudan-TSI/Human_Practices)
  - [Collaboration](https://2019.igem.org/Team%3AFudan-TSI/Collaborations)
  - [Safety](https://2019.igem.org/Team%3AFudan-TSI/Safety)
+ [Team](https://2019.igem.org/Team%3AFudan-TSI/Team)

  - [Team Members](https://2019.igem.org/Team%3AFudan-TSI/Team)
  - [Attribution](https://2019.igem.org/Team%3AFudan-TSI/Attributions)
  - [Acknowledgement](https://2019.igem.org/Team%3AFudan-TSI/Acknowledgment)
+ [Judging](https://2019.igem.org/Team%3AFudan-TSI/Judging)

![](https://static.igem.org/mediawiki/2019/a/a3/T--Fudan-TSI--coverResults.gif)

* [Transcription](#mainTitle1)
* [Reverse transcription](#mainTitle2)
  + [The successful expression of reverse transcriptase](#mainTitle2_1)
* [Recombination](#mainTitle3)
  + [Cre expression is controlled by Tet operon](#mainTitle3_1)
  + [Cre initiates excision between two homologous loxP site](#mainTitle3_2)
  + [lox5171 is most incompatible with wildtype loxP (wtlox)](#mainTitle3_3)
  + [Cre with degradation tags](#mainTitle3_4)
* [System verification](#mainTitle4)
* [References](#mainTitle5)

Our system can be divided into three biochemical steps: transcription of the target sequence into mRNA, reverse transcription of the target sequence into double-stranded cDNA, and recombination for the cDNA to replace the target sequence. We set out to separately verify each step.

Transcription

We put the target sequence and its flanking elements together under a T7 stable promoter for high expression level of target RNA. To verify our R-Evolution system, we constructed 8 nonsense mutant of chloramphenicol resistance gene (Chl), bearing the 8 base pair substitution from sense codon to nonsense mutant. We verified this construct through culturing bacteria carrying the original version or mutant on plates containing chloramphenicol. We found that bacteria carrying the original Chl grow naturally, while no colony was formed on the plates of mChl (Table. 1). After adding the flanking sequences on both ends, we used the C158X mutant and showed that the gene’s function has not been changed by our conduct (Fig. 1).

|  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Control | Chl | Chl-Y33X | Chl-W85X | Chl-E97X | Chl-S121X | Chl-L158X | Chl-K182X | Chl-Q190X |
| Amp | 19 | 192 | 291 | 128 | 236 | 138 | 255 | 175 | 276 |
| Amp & Chl | 0 | 30 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |

![]()

**Table 1. Nonsense mutation disrupts the chloramphenicol resistance ability of Chl.**
Amp stands for ampicillin, Chl stands for chloramphenicol, the left column means that they are added to the plate. Chl acts as the positive control, cells grow naturally on both plates, while cells transformed with the negative control plasmid can only grow on plates containing ampicillin. Chl gene carrying nonsense mutation at different sites does not affect its ampicillin resistance, but chloramphenicol resistance is lost as no colony is formed. Single colony was picked and shook in liquid culture overnight, the plate was coated with 100 μL culture after 104 dilution.

![](https://static.igem.org/mediawiki/2019/4/4b/T--Fudan-TSI--Result_Fig1.gif)

**Figure 1. The flanking of reverse transcription initiation and loxP sequences does not disrupt Chl functionality.**

In addition, we also constructed and verified nonsense mutants of fluorescent protein EGFP and mCherry at the 158th and 159th amino acid (Fig. 2), based on the split EGFP assay that either half of the protein cannot emit fluorescence1.

![](https://static.igem.org/mediawiki/2019/4/45/T--Fudan-TSI--Result_Fig2.gif)

**Figure 2. Nonsense mutant of EGFP and mCherry cannot emit fluorescence.**

Reverse transcription

The successful expression of reverse transcriptase

RT is expressed under an IPTG controlled promoter, we constructed a series of promoters by placing a LacO fragment under a stable promoter, and hopes to determine under which we could achieve most stringent control. The constructs we tested are—T5, T5-LacO, T7-LacO, LacUV5-LacO, J23119-LacO.

We initially attempted to verify RT’s expression through an EGFP fusion protein. We fused the EGFP to the C’ end of pol protein, linked by a GS tag. But this construct proved unsuccessful (data not shown), possibly due to the length and complexity of the gag-pol polyprotein. Then we turned to directly expressing EGFP in the place of RT under the control of IPTG. If EGFP can be successfully expressed, so should RT. And results are obtained for each induction promoter construct (figure would be supplemented in presentation and poster).

We found through careful examination that this failure is due to problems with our plasmid construct, so we moved the RT to another tested plasmid, and through SDS-PAGE, verified its successful expression (Fig. 3).

![](https://static.igem.org/mediawiki/2019/2/2f/T--Fudan-TSI--Result_Fig3.gif)

**Figure 3. gag-pol polyprotein is successfully expressed and underwent excision in the cell.**

Recombination

Cre expression is controlled by Tet operon

We placed Cre under the promoter ptetR, whose expression is controlled by its inhibitor TetR. Regarding where we should place the inhibitor gene to maximize its expression, we opted between 2 options, one is placing it downstream of the LacI inhibitor, the other is to place it downstream of the kanamycin resistance gene (KanR). We tested both construct by placing an EGFP in these two places and measuring its fluorescence emission. Results show that the expression level is almost the same in both construct (Fig. 4).

![](https://static.igem.org/mediawiki/2019/2/2f/T--Fudan-TSI--Result_Fig4.gif)

**Figure 4. EGFP expression level is relatively the same downstream of LacI or KanR.**

Cre initiates excision between two homologous loxP site

Placing 2 wild-type loxP on both ends of the target sequence (mCherry) in the same direction, and expressing it under a stable promoter (J23101). By co-transforming the target plasmid with another plasmid carrying Cre recombinase, we verified that our Cre protein functions accordingly by excising the mCherry sequence from the promoter (Fig. 5). Through PCR amplification with the primers annealing to sequences outside the target, and subsequent electrophoresis, we found that the band from bacteria co-transforming Cre corresponds to the excision of mCherry.

![](https://static.igem.org/mediawiki/2019/e/e8/T--Fudan-TSI--Result_Fig5.gif)

**Figure 5. lox511 remains compatible with wildtype loxP, though at a lower excision rate.**
Wildtype loxP and lox511-mCherry-loxP are analyzed on two different gels, their marker bands are indicated. Wildtype loxP only has an excision band. lox511 has a slight full-length mCherry band slightly longer than 1000 bp, which corresponds with the Ctr+ result in Fig. 6, but excision band is still visible and brighter than that of full-length mCherry. This result suggests that lox511 still interacts with wildtype loxP and go through excision, but at a lower efficiency.

lox5171 is most incompatible with wildtype loxP (wtlox)

When we were carrying out integrated human practice, we were warned by Prof. Wang that two homologous loxP would be excised at a much higher efficiency than performing recombination as we wished, so we searched the literature and selected 3 mutants that are said to be incompatible with wt-loxP but are compatible with themselves, they are lox511, lox2272 and lox5171. We tested their incompatibility with wt-loxP by replacing one of wt-loxP into the mutant at the ends of mCherry, and co-transformed the plasmid with Cre (Fig. 5 & 7). The result we obtained showed that lox5171-mCherry-wt\_loxP performs best, and used it in further analysis (Fig. 7).

![](https://static.igem.org/mediawiki/2019/9/9e/T--Fudan-TSI--Result_Fig6.gif)

**Figure 6. Schematic diagram of loxP mutant incompatibility test.**

![](https://static.igem.org/mediawiki/2019/b/b3/T--Fudan-TSI--Result_Fig7.gif)

**Figure 7. Cre excises sequences flanked by homologous loxP sites, but are incompatible with its mutant version.**
The above column shows which plasmids are transformed. The 3 middle lanes stand for Cre co-transforming with mCherry flanked on both ends by wildtype loxP (Lane 3), or with wildtype loxP on only one end, the other end being lox2272 (Lane 4) or lox5171 (Lane 5). mCherry flanked with lox2272 or lox5171 on one end does not go through excision so a full-length band was detectable, while mCherry flanked with wildtype loxP on both ends are excised and only a shorter band was seen.

Cre with degradation tags

When our modelling demonstrated to us that the expression level of Cre needs to be much lower than that of RT, we introduced degradation tags. By attaching them to the C terminal of Cre recombinase, the protein would be rapidly recognized and degraded by the E. coli’s native SsrA-SmpB degradation system. This construct could also solve the problem of basal leakage and continued existence after inducer removal. Apart from the native AANDENYALAA tag, we also modified its last three or five amino acids into YALAV, YALVA, YALVV and WVLAA. We tested the stable expression level of each tag by attaching them to the C terminal of EGFP protein and measuring the change in fluorescence level (Fig. 8). The stable state expression increases as the number of mutated amino acids increase, or the mutated site nears the N’ of the tag. Supported by our modelling result, we deemed that the WVLAA tag performs best and chose to use it in further experiments.

![](https://static.igem.org/mediawiki/2019/6/61/T--Fudan-TSI--Result_Fig8.gif)

**Figure 8. Degradation tag greatly reduces the protein level at stable state.**
WT represents the positive control of EGFP without any tag attachment. The five degradation tags are represented by their last five amino acid sequence. The vertical axis shows the quantitative analysis of EGFP fluorescence (excitation wavelength: 485 nm; detection wavelength: 528 nm), normalized by cell amount (OD600). The fluorescence is quantified by the concentration of green fluorescein, cell number is quantified by the number of silicon beads, both are from the distributed measurement kit. Fluorescence below detection level are eliminated. Error bar stands for the SEM of 3 replicates. t-test is performed between WT and each degradation tag, P<0.0001 (\*\*\*\*).

System verification

We would test the system through both the recovery of EGFP fluorescence of its nonsense mutant, and the recovery of chloramphenicol resistance from its nonsense mutation. Fluorescence would provide to us a quick assessment of our system’s efficiency, while Chl resistance could offer a quantitative measurement of the reversion rate.
Due to lack of time, we have not finished our system verification experiment. But we represent here the anticipated result that should be obtained.

References

* 1. [Pathmanathan, S., Barnard, E. & Timson, D. J. Interactions between the budding yeast IQGAP homologue Iqg1p and its targets revealed by a split-EGFP bimolecular fluorescence complementation assay. *Cell Biol. Int.* 32, 1318–1322 (2008).](https://onlinelibrary.wiley.com/doi/abs/10.1016/j.cellbi.2008.07.008)

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### R-Evolution: an *in vivo* sequence-specific toolbox for continuous mutagenesis

[Project](/Team%3AFudan-TSI/Description)

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