iGEM 2021 — Team OUC-R

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:OUC-R/Notebook

[![](https://static.igem.org/mediawiki/2021/f/fe/T--OUC-R--logo_.png)](https://2021.igem.org/Team%3AOUC-R)

* PROJECT
  + [Description](https://2021.igem.org/Team%3AOUC-R/Description)
  + [Design](https://2021.igem.org/Team%3AOUC-R/Design)
  + [Results](https://2021.igem.org/Team%3AOUC-R/Results)
  + [Contribution](https://2021.igem.org/Team%3AOUC-R/Contribution)
  + [Engineering](https://2021.igem.org/Team%3AOUC-R/Engineering)
  + [Implementation](https://2021.igem.org/Team%3AOUC-R/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AOUC-R/Proof_Of_Concept)
  + [Medal Equirement](https://2021.igem.org/Team%3AOUC-R/Medal_Equirement)
* WET LAB
  + [Experiments](https://2021.igem.org/Team%3AOUC-R/Experiments)
  + [Safety](https://2021.igem.org/Team%3AOUC-R/Safety)
  + [Measurement](https://2021.igem.org/Team%3AOUC-R/Measurement)
* [MODEL](https://2021.igem.org/Team%3AOUC-R/Model)
  + [Overview](https://2021.igem.org/Team%3AOUC-R/Model)
  + [Sequence Design](https://2021.igem.org/Team%3AOUC-R/Model/sd)
  + [ODE](https://2021.igem.org/Team%3AOUC-R/Model/ODE)
  + [Furthermore](https://2021.igem.org/Team%3AOUC-R/Model/Futh)
  + [Software](https://2021.igem.org/Team%3AOUC-R/Software)
* [PARTS](https://2021.igem.org/Team%3AOUC-R/Parts)
  + [Overview](https://2021.igem.org/Team%3AOUC-R/Parts)
  + [Improvement](https://2021.igem.org/Team%3AOUC-R/Parts#part_impro)
* [HUMAN PRACTICES](https://2021.igem.org/Team%3AOUC-R/Human_Practices)
  + [Human Practices](https://2021.igem.org/Team%3AOUC-R/Human_Practices)
  + [Education](https://2021.igem.org/Team%3AOUC-R/Education)
  + [Online Lectures](https://2021.igem.org/Team%3AOUC-R/Education#Online_Lectures )
  + [Comics](https://2021.igem.org/Team%3AOUC-R/Education#Comics)
  + [Scientific Videos](https://2021.igem.org/Team%3AOUC-R/Education#Scientific_Videos)
  + [Communication](https://2021.igem.org/Team%3AOUC-R/Communication)
* [COLLABORATIONS](https://2021.igem.org/Team%3AOUC-R/Collaborations)
  + [Collaborations](https://2021.igem.org/Team%3AOUC-R/Collaborations)
  + [OUC-China\_Team](https://2021.igem.org/Team%3AOUC-R/Collaborations#OUC-CHINA Team)
  + [Tongji-Software Team](https://2021.igem.org/Team%3AOUC-R/Collaborations#Tongji-Software_Team)
  + [ICJFLS Team](https://2021.igem.org/Team%3AOUC-R/Collaborations#ICJFLS_Team)
  + [ECUST\_China Team](https://2021.igem.org/Team%3AOUC-R/Collaborations#ECUST_China_Team)
* [TEAM](https://2021.igem.org/Team%3AOUC-R/Team)
  + [Members](https://2021.igem.org/Team%3AOUC-R/Team)
  + [Attributions](https://2021.igem.org/Team%3AOUC-R/Attributions)

[![](https://static.igem.org/mediawiki/2021/b/b0/T--OUC-R--totop.png)](#toptop)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:OUC-R/Engineering

[![](https://static.igem.org/mediawiki/2021/f/fe/T--OUC-R--logo_.png)](https://2021.igem.org/Team%3AOUC-R)

* PROJECT
  + [Description](https://2021.igem.org/Team%3AOUC-R/Description)
  + [Design](https://2021.igem.org/Team%3AOUC-R/Design)
  + [Results](https://2021.igem.org/Team%3AOUC-R/Results)
  + [Contribution](https://2021.igem.org/Team%3AOUC-R/Contribution)
  + [Engineering](https://2021.igem.org/Team%3AOUC-R/Engineering)
  + [Implementation](https://2021.igem.org/Team%3AOUC-R/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AOUC-R/Proof_Of_Concept)
  + [Medal Equirement](https://2021.igem.org/Team%3AOUC-R/Medal_Equirement)
* WET LAB
  + [Experiments](https://2021.igem.org/Team%3AOUC-R/Experiments)
  + [Safety](https://2021.igem.org/Team%3AOUC-R/Safety)
  + [Measurement](https://2021.igem.org/Team%3AOUC-R/Measurement)
* [MODEL](https://2021.igem.org/Team%3AOUC-R/Model)
  + [Overview](https://2021.igem.org/Team%3AOUC-R/Model)
  + [Sequence Design](https://2021.igem.org/Team%3AOUC-R/Model/sd)
  + [ODE](https://2021.igem.org/Team%3AOUC-R/Model/ODE)
  + [Furthermore](https://2021.igem.org/Team%3AOUC-R/Model/Futh)
  + [Software](https://2021.igem.org/Team%3AOUC-R/Software)
* [PARTS](https://2021.igem.org/Team%3AOUC-R/Parts)
  + [Overview](https://2021.igem.org/Team%3AOUC-R/Parts)
  + [Improvement](https://2021.igem.org/Team%3AOUC-R/Parts#part_impro)
* [HUMAN PRACTICES](https://2021.igem.org/Team%3AOUC-R/Human_Practices)
  + [Human Practices](https://2021.igem.org/Team%3AOUC-R/Human_Practices)
  + [Education](https://2021.igem.org/Team%3AOUC-R/Education)
  + [Online Lectures](https://2021.igem.org/Team%3AOUC-R/Education#Online_Lectures )
  + [Comics](https://2021.igem.org/Team%3AOUC-R/Education#Comics)
  + [Scientific Videos](https://2021.igem.org/Team%3AOUC-R/Education#Scientific_Videos)
  + [Communication](https://2021.igem.org/Team%3AOUC-R/Communication)
* [COLLABORATIONS](https://2021.igem.org/Team%3AOUC-R/Collaborations)
  + [Collaborations](https://2021.igem.org/Team%3AOUC-R/Collaborations)
  + [OUC-China\_Team](https://2021.igem.org/Team%3AOUC-R/Collaborations#OUC-CHINA Team)
  + [Tongji-Software Team](https://2021.igem.org/Team%3AOUC-R/Collaborations#Tongji-Software_Team)
  + [ICJFLS Team](https://2021.igem.org/Team%3AOUC-R/Collaborations#ICJFLS_Team)
  + [ECUST\_China Team](https://2021.igem.org/Team%3AOUC-R/Collaborations#ECUST_China_Team)
* [TEAM](https://2021.igem.org/Team%3AOUC-R/Team)
  + [Members](https://2021.igem.org/Team%3AOUC-R/Team)
  + [Attributions](https://2021.igem.org/Team%3AOUC-R/Attributions)

[![](https://static.igem.org/mediawiki/2021/b/b0/T--OUC-R--totop.png)](#toptop)

![](https://static.igem.org/mediawiki/2021/5/55/T--OUC-R--EGNEW.png)

# Engineering

In this section, we will introduce our engineering design cycle.We designed and constructed the first model containing APP sequences, and then optimized the experimental conditions to improve its efficiency.Later, we designed other different models, such as the model without APP sequence, and explored the regulation of regulatory factors on them through experiments.

## The engineering cycle

Research→Imagine→Design→Build→Test→Improve→Test→Design→Test→Future

## Research：How to regulate transcription by b-DNA and Z-DNA conversion?

To understand the mechanism of conversion between B-DNA and Z-DNA, we reviewed the literature and sought the help of relevant experts.We found that the APP sequence is essencial for conversion between the two, and the conversion between B-DNA and Z-DNA can be achieved by adding ZBP1 antibody, spermatic ammonia ion and other regulatory factors.On the premise of keeping the chain number unchanged, the conversion of B-DNA to Z-DNA can open the hairpin structure, so that DNA polymerase can easily combine with the promoter sequence in the hairpin structure, and realize the regulation of transcription.

## Imagine: How to testify the regulatory effects?

We hope to use the nuclear enzyme to detect our regulatory effects. We think that if we can regulate transcription correctly, then we transcribe the nuclease. We use the existing RNA to verify that the transcription produces the product we want to get -- the nuclease. If the cutting is successful, it can prove our regulatory effect.

## Design: How to design a model containing APP sequence?

We designed a circular double stranded DNA model with a cross structure，with part of the promoter subsequence contained in the cross structure。We add the app sequence in the front end of the cross structure, hoping that the subsequent experiment will change the structure of the app sequence to achieve the purpose of transcription. And the sequence after the promoter is the DNA sequence of the ribozyme that we want to express. The specific design can be checked from [design](https://2021.igem.org/Team%3AOUC-R/Design) part.

## Build：How to build a model?

After completing the design of the model sequence, we ordered from the company our sequence needed. We also ordered a splint chain to help our designed DNA sequences to form a cycle, and we build our model with these DNA chains. We divide the model into three parts，including the cyclization of single stranded DNA、the preparation of the double loop topology isomers and transcriptional regulation.

## Test: Are we getting the desired results?

We constructed our model through the experiment, and in each part of the model, we conducted the preliminary experimental results by electrophoresis, and analyzed our results through the gel imager to calculate the efficiency of our experiment. Please check our results page for the specific [results](https://2021.igem.org/Team%3AOUC-R/Results).

## Improve: How to improve our experimental plan?

We found that in the initial reaction conditions, the substrate had more surplus and the reaction efficiency was not very high. In order to improve the yield, we set up different reaction conditions in the experiment to explore the optimum conditions of our experiment. In addition, we have improved our experimental methods to obtain the products we want to get.

## Test: Verify the improved experimental scheme

Again, we tested the experimental conditions and the scheme of our improvement. After the electrophoresis and coagulation imaging analysis, we chose our final experimental program in many experimental conditions and conducted experiments in that very experimental conditions.

## Design: design other different models

On the basis of the model that contains the app sequence, we designed other different models to better validate our results. Other models we design include one without the app sequence, one without the cross structure, and one that contains a model that does not match the cross structure.

## Test: to verify different models with experiments

In order to better verify our results, we build and experiment with other models and compare the results of the experiment to verify whether our reasoning is correct.

## Future: Our expectations

We have designed an experiment in vitro. In the future, we hope to use plasmids to import our models into microbial cells, allowing them to regulate transcription in cells to regulate the expression of genetic material.

![](https://static.igem.org/mediawiki/2021/5/5e/T--OUC-R--BANQAUN.png)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:OUC-R/Experiments

[![](https://static.igem.org/mediawiki/2021/f/fe/T--OUC-R--logo_.png)](https://2021.igem.org/Team%3AOUC-R)

* PROJECT
  + [Description](https://2021.igem.org/Team%3AOUC-R/Description)
  + [Design](https://2021.igem.org/Team%3AOUC-R/Design)
  + [Results](https://2021.igem.org/Team%3AOUC-R/Results)
  + [Contribution](https://2021.igem.org/Team%3AOUC-R/Contribution)
  + [Engineering](https://2021.igem.org/Team%3AOUC-R/Engineering)
  + [Implementation](https://2021.igem.org/Team%3AOUC-R/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AOUC-R/Proof_Of_Concept)
  + [Medal Equirement](https://2021.igem.org/Team%3AOUC-R/Medal_Equirement)
* WET LAB
  + [Experiments](https://2021.igem.org/Team%3AOUC-R/Experiments)
  + [Safety](https://2021.igem.org/Team%3AOUC-R/Safety)
  + [Measurement](https://2021.igem.org/Team%3AOUC-R/Measurement)
* [MODEL](https://2021.igem.org/Team%3AOUC-R/Model)
  + [Overview](https://2021.igem.org/Team%3AOUC-R/Model)
  + [Sequence Design](https://2021.igem.org/Team%3AOUC-R/Model/sd)
  + [ODE](https://2021.igem.org/Team%3AOUC-R/Model/ODE)
  + [Furthermore](https://2021.igem.org/Team%3AOUC-R/Model/Futh)
  + [Software](https://2021.igem.org/Team%3AOUC-R/Software)
* [PARTS](https://2021.igem.org/Team%3AOUC-R/Parts)
  + [Overview](https://2021.igem.org/Team%3AOUC-R/Parts)
  + [Improvement](https://2021.igem.org/Team%3AOUC-R/Parts#part_impro)
* [HUMAN PRACTICES](https://2021.igem.org/Team%3AOUC-R/Human_Practices)
  + [Human Practices](https://2021.igem.org/Team%3AOUC-R/Human_Practices)
  + [Education](https://2021.igem.org/Team%3AOUC-R/Education)
  + [Online Lectures](https://2021.igem.org/Team%3AOUC-R/Education#Online_Lectures )
  + [Comics](https://2021.igem.org/Team%3AOUC-R/Education#Comics)
  + [Scientific Videos](https://2021.igem.org/Team%3AOUC-R/Education#Scientific_Videos)
  + [Communication](https://2021.igem.org/Team%3AOUC-R/Communication)
* [COLLABORATIONS](https://2021.igem.org/Team%3AOUC-R/Collaborations)
  + [Collaborations](https://2021.igem.org/Team%3AOUC-R/Collaborations)
  + [OUC-China\_Team](https://2021.igem.org/Team%3AOUC-R/Collaborations#OUC-CHINA Team)
  + [Tongji-Software Team](https://2021.igem.org/Team%3AOUC-R/Collaborations#Tongji-Software_Team)
  + [ICJFLS Team](https://2021.igem.org/Team%3AOUC-R/Collaborations#ICJFLS_Team)
  + [ECUST\_China Team](https://2021.igem.org/Team%3AOUC-R/Collaborations#ECUST_China_Team)
* [TEAM](https://2021.igem.org/Team%3AOUC-R/Team)
  + [Members](https://2021.igem.org/Team%3AOUC-R/Team)
  + [Attributions](https://2021.igem.org/Team%3AOUC-R/Attributions)

[![](https://static.igem.org/mediawiki/2021/b/b0/T--OUC-R--totop.png)](#toptop)

![](https://static.igem.org/mediawiki/2021/d/d3/T--OUC-R--SSSB1.png)

# Experiments

## Phosphorylation (20μM) [Click To show The Details]

DNA samples

PNK

ATP

H2O

buffer：500mM Tris-HCl(pH7.6 at 25℃)、100mM MgCl2、50mM DTT、1mM spermidine.

## Preparation of CT1 /CS2 (1μM) [Click To show The Details]

Phosphorylated sample (20μM)

T4 ligase

H2O

buffer：400mM Tris-HCl、100mM MgCl2、100mM DTT、5mM ATP(pH7.8 at 25℃)

## Preparation of ET1/ ES2 (0.75μM) [Click To show The Details]

CT1/CS2 (1μM)

Exo I

H2O

buffer：670mM glycine-KOH(pH9.5 at 25℃)、67mM MgCl2、10mM DTT

## Double circle DNA cyclization [Click To show The Details]

ET1/ ES2(0.75μM)

S1P1、S1P2/T2P1、T2P2

T4 ligase

H2O

buffer：400mM Tris-HCl、100mM MgCl2、100mM DTT、5mM ATP(pH7.8 at 25℃)

## Double circle DNA digestion [Click To show The Details]

Cyclized DNA sample (see protocol cyclization)

Exo I

Exo III

buffer1：670mM glycine-KOH (pH9.5 at 25℃)、67mM MgCl2、10mM DTT

buffer2：660mM Tris-HCl (pH8.0 at 30℃)、6.6mM MgCl2

## Transcription [Click To show The Details]

digested DNA sample (see protocol digestion)

NTP

T7 RNA polymerase

H2O

RNA Degradation inhibitor

Z22/ZBP1(Added in Transcriptional regulation experiment)

buffer：200mM Tris-HCl(pH8.0)、40mM MgCl2、10mM spermidine -(HCl)3、125mM NaCl

![](https://static.igem.org/mediawiki/2021/5/5e/T--OUC-R--BANQAUN.png)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:OUC-R/Results

[![](https://static.igem.org/mediawiki/2021/f/fe/T--OUC-R--logo_.png)](https://2021.igem.org/Team%3AOUC-R)

* PROJECT
  + [Description](https://2021.igem.org/Team%3AOUC-R/Description)
  + [Design](https://2021.igem.org/Team%3AOUC-R/Design)
  + [Results](https://2021.igem.org/Team%3AOUC-R/Results)
  + [Contribution](https://2021.igem.org/Team%3AOUC-R/Contribution)
  + [Engineering](https://2021.igem.org/Team%3AOUC-R/Engineering)
  + [Implementation](https://2021.igem.org/Team%3AOUC-R/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AOUC-R/Proof_Of_Concept)
  + [Medal Equirement](https://2021.igem.org/Team%3AOUC-R/Medal_Equirement)
* WET LAB
  + [Experiments](https://2021.igem.org/Team%3AOUC-R/Experiments)
  + [Safety](https://2021.igem.org/Team%3AOUC-R/Safety)
  + [Measurement](https://2021.igem.org/Team%3AOUC-R/Measurement)
* [MODEL](https://2021.igem.org/Team%3AOUC-R/Model)
  + [Overview](https://2021.igem.org/Team%3AOUC-R/Model)
  + [Sequence Design](https://2021.igem.org/Team%3AOUC-R/Model/sd)
  + [ODE](https://2021.igem.org/Team%3AOUC-R/Model/ODE)
  + [Furthermore](https://2021.igem.org/Team%3AOUC-R/Model/Futh)
  + [Software](https://2021.igem.org/Team%3AOUC-R/Software)
* [PARTS](https://2021.igem.org/Team%3AOUC-R/Parts)
  + [Overview](https://2021.igem.org/Team%3AOUC-R/Parts)
  + [Improvement](https://2021.igem.org/Team%3AOUC-R/Parts#part_impro)
* [HUMAN PRACTICES](https://2021.igem.org/Team%3AOUC-R/Human_Practices)
  + [Human Practices](https://2021.igem.org/Team%3AOUC-R/Human_Practices)
  + [Education](https://2021.igem.org/Team%3AOUC-R/Education)
  + [Online Lectures](https://2021.igem.org/Team%3AOUC-R/Education#Online_Lectures )
  + [Comics](https://2021.igem.org/Team%3AOUC-R/Education#Comics)
  + [Scientific Videos](https://2021.igem.org/Team%3AOUC-R/Education#Scientific_Videos)
  + [Communication](https://2021.igem.org/Team%3AOUC-R/Communication)
* [COLLABORATIONS](https://2021.igem.org/Team%3AOUC-R/Collaborations)
  + [Collaborations](https://2021.igem.org/Team%3AOUC-R/Collaborations)
  + [OUC-China\_Team](https://2021.igem.org/Team%3AOUC-R/Collaborations#OUC-CHINA Team)
  + [Tongji-Software Team](https://2021.igem.org/Team%3AOUC-R/Collaborations#Tongji-Software_Team)
  + [ICJFLS Team](https://2021.igem.org/Team%3AOUC-R/Collaborations#ICJFLS_Team)
  + [ECUST\_China Team](https://2021.igem.org/Team%3AOUC-R/Collaborations#ECUST_China_Team)
* [TEAM](https://2021.igem.org/Team%3AOUC-R/Team)
  + [Members](https://2021.igem.org/Team%3AOUC-R/Team)
  + [Attributions](https://2021.igem.org/Team%3AOUC-R/Attributions)

[![](https://static.igem.org/mediawiki/2021/b/b0/T--OUC-R--totop.png)](#toptop)

![](https://static.igem.org/mediawiki/2021/8/8b/T--OUC-R--r1.png)

# Results

![](https://static.igem.org/mediawiki/2021/1/1c/T--OUC-R--RR1.png)

![](https://static.igem.org/mediawiki/2021/3/32/T--OUC-R--RR2.png)

At the beginning of the experiment, we phosphorylated the single strand of DNA, that is, ATP provided the phosphate group, and PNK enzyme added phosphoric acid to the 5 'end of the single strand for subsequent loop formation.

Next, we added the T4 ligase to make the long single strand of DNA into a loop, and in this process, we cycled T1 and S2 in different ways. T1 uses its own hairpin structure to fix the two ends that need to be looped, and then lets T4 ligase repair the gap to make it looped. For S2, we add the auxiliary splint chain to fix both ends of the chain and increase the probability of loop formation.

At the same time, we also tried to use two short chains to connect a single ring structure, the main purpose of this is to reduce the economic cost. The single strand that was not connected to the loop and the splint strand were removed by ExoI after enzyme digestion. Thus, we have T1 and S2 monocycles with almost no impurities.

Hint: the abbreviation P stands for phosphorylation and the word C stands for cyclized and d stands for digested.

## Long chain connection:

![](https://static.igem.org/mediawiki/2021/b/b7/T--OUC-R--1.jpg)

![](https://static.igem.org/mediawiki/2021/f/f0/T--OUC-R--2.jpg)

## Short chain connection:

![](https://static.igem.org/mediawiki/2021/a/ae/T--OUC-R--s3.jpg)

![](https://static.igem.org/mediawiki/2021/0/0a/T--OUC-R--s4.jpg)

![](https://static.igem.org/mediawiki/2021/6/66/T--OUC-R--5.jpg)

![](https://static.igem.org/mediawiki/2021/1/11/T--OUC-R--6.jpg)

![](https://static.igem.org/mediawiki/2021/2/2e/T--OUC-R--7.jpg)

![](https://static.igem.org/mediawiki/2021/e/e7/T--OUC-R--new8.jpg)

![](https://static.igem.org/mediawiki/2021/0/05/T--OUC-R--new9.jpg)

![](https://static.igem.org/mediawiki/2021/d/d1/T--OUC-R--10.jpg)

In order to better obtain the double ring structure, we adopt the method of two connected segments. The so-called two-segment link refers to a single ring and two short chains connected to form a double ring structure. The results show that this method can get the desired structure. In this step, we first put T1 single ring and S1P2 in the same system, and then put S1P1 in the other system, after annealing and denaturing together, add enough enzyme to S1P1 and mix the two systems quickly. Finally, put it into the appropriate temperature and react for 2 hours. Similarly, short chains T2P1 and T2P2 were added to S2 in the same way to form a double-ring structure.

![](https://static.igem.org/mediawiki/2021/8/83/T--OUC-R--SB1.png)

![](https://static.igem.org/mediawiki/2021/5/52/T--OUC-R--SB2.png)

![](https://static.igem.org/mediawiki/2021/a/a5/T--OUC-R--XIUGAI1.png)

After that, double ring enzymatic digestion was started, which was similar to single ring enzymatic digestion. In order to improve the purity of the product, exoIII with better effect was added, and the reaction was carried out at 37℃ for two hours, and the double ring structure was obtained.

**It is speculated that Lk7 (δ LK-1) was prepared from channel 8, which may contain a small amount of Lk6 (δ LK-2) and Lk5 (δ LK-3). Lane 14 also has a small amount of Lk7. With the presence of topological limiting forces, Lk7 is easy to form.**

![](https://static.igem.org/mediawiki/2021/1/18/T--OUC-R--11.png)

![](https://static.igem.org/mediawiki/2021/7/75/T--OUC-R--12new.png)

![](https://static.igem.org/mediawiki/2021/8/86/T--OUC-R--13.png)

![](https://static.igem.org/mediawiki/2021/9/9b/T--OUC-R--14.png)

![](https://static.igem.org/mediawiki/2021/5/5a/T--OUC-R--16.png)

![](https://static.igem.org/mediawiki/2021/2/25/T--OUC-R--17.png)

In the end we transcribe.

In the transcription results without inducers, it can be seen that APP sequence and topological isomers with cross structure can promote transcription under experimental conditions, which may be because z-DNA structure is easy to form in the presence of high concentration of magnesium ions, and cross structure is opened at the same time. Of course, there is another situation, that is, a bubble structure is formed in the double ring structure, which makes the enzyme easy to bind to the bubble structure, thus making the initiation of transcription easier, what’s improving the efficiency of transcription.

![](https://static.igem.org/mediawiki/2021/0/0e/T--OUC-R--18.png)

![](https://static.igem.org/mediawiki/2021/3/3d/T--OUC-R--19.png)

From the results of adding Z22 protein and ZBP-1 protein for transcription, it can be seen that the added Z22 protein and ZBP-1 protein can inhibit gene transcription, and the inhibition effect of ZBP-1 is stronger than that of Z22. Although there is a certain gap between this and the expected experimental results, we will continue to improve our experiment, adjust the experimental conditions, To further explore the effects of left and right helix and cross structure on gene expression.

![](https://static.igem.org/mediawiki/2021/8/81/T--OUC-R--20.png)

![](https://static.igem.org/mediawiki/2021/e/e6/T--OUC-R--21.png)

![](https://static.igem.org/mediawiki/2021/5/5e/T--OUC-R--BANQAUN.png)