iGEM 2021 — Team XJTU-China

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

# Team:XJTU-China/Notebook

1. sideMenu, #top\_title, .patrollink, #firstHeading, #home\_logo, #sideMenu,a#top,p:empty, #top\_menu\_under { display:none; }
2. content { padding:0px; width:100%; margin-top:-7px; margin-left:0px; border:none;}
3. bodyContent h1, #bodyContent h2, #bodyContent h3, #bodyContent h4, #bodyContent h5 { margin-bottom: 0px; }
4. bodyContent a[href ^="https://"], .link-https { padding-right:0px;}
5. globalWrapper {padding-bottom:0}

# 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:XJTU-China/Engineering

Team:XJTU-China/Engineering

Engineering

### Engineering

* [1. General Design](#1)
* [2. *In
  silico*
           Engineering](#2)
  + [2.1 Design, Build and Test](#2.1)
  + [2.2 Learn](#2.2)
* [3.
  Experimental
           Engineering](#3)
  + [3.1 Design](#3.1)
  + [3.2 Build](#3.2)
  + [3.3 Test](#3.3)
    - [3.3.1 RT-qPCR](#3.3.1)
    - [3.3.2 Characterization](#3.3.2)
  + [3.4 Learn](#3.4)

# Engineering Success Toggle Switch

## 1. General Design

This circuit is an updated version of the traditional toggle switch
developed by James J.
Collins's group in 2000 (Gardner et al. Nature, 2000), which contains two
feedback each
controls the other, and can achieve bistability of protein expression in
different
inducing conditions.

Two promoter-repressor systems (lacUV5 promoter-lacI and lambda pomoter-CI857) with *sfGFP*
and
mRFP respectively, was constructed in our updated version, to monitor the two
states of
the circuit. With induction of IPTG, the downstream genes of lacUV5, that is, CI
protein
and *mRFP* will expressed, while those in the downstream of lambda promoter (*lacI*
and
*sfGFP*) will be repressed. Even without IPTG induction after several hours, the
lack of
*lacI* expression will result in the stability of red fluorescence. At
temperatures above
42 ℃, gene expression will be flipped into another state, the
stable
expression of *lacI* and *sfGFP*, and the state will maintain even without heat. The
GFP and
RFP can be altered with other functional genes such as tyrptohan synthesitic
genes to
achieve the bistable expression and synthesis of tyrptohan.

The design of toggle-switch circuit is shown in Fig. 1.1.

![Design of Toggle-Switch Circuit](https://static.igem.org/mediawiki/2021/8/81/T--XJTU-China--ES.Fig.1.1.png)
**Fig. 1.1** Design of Toggle-Switch
Circuit

## 2. *in-silico* Engineering

### 2.1 Design, Build and Test

Before our experiment, modeling was performed for
*in-silico*
prediction
of behaviours of the toggle switch circuit. We constructed a equation set
based on
transcription and expression to describe the changes in the relative
expression
levels of GFP and RFP under different inducing conditions (IPTG induction or
42℃
heat). Equations used are listed in Fig. 2.1

The *in silico* result of the expression level of *sfGFP* and
*mRFP* under
different leaky expression factor α0 as shown in
Fig2.2， which indicates
that the behavior of toggle switch is largely affected by the leaky
expression of two promoters. A bistability can be achieved under low α0,
and toggle switch will fail in case of high α0.

![the model of toggle switch](https://static.igem.org/mediawiki/2021/e/e9/T--XJTU-China--result_2.png)
**Fig. 2.2 Toggle Switch Modelling
Result.**
IPTG is
introduced at 1000 min
and the induction condition is switched to 42℃ at 2000 min.

![the result of toggle switch modelling](https://static.igem.org/mediawiki/2021/2/24/T--XJTU-China--engineering-equations.png)
**Fig. 2.1 Toggle Switch Modelling
Equations.**

For detail, see also our[Modelling](https://2021.igem.org/Team%3AXJTU-China/Model)

### 2.2 Learn

With the result of our *in-silico* prediction, we concluded that
reduced
leaky expression of promoters is the key to construct the toggle-switch circuit.
This would provide guidance for our following experimental design, including
selection of the RBSs with appropriate strength to weaken the possible leaky
expressions.

## 3. Experimental Engineering

### 3.1 Design

Generally, the toggle-switch circuit is constructed utilizing two sets
of
promoter-repressor system, namely, lacUV5 promoter-LacI and lambda promoter-CI857,
in
addition to *sfGFP* and *mRFP* as the reporter genes (see the Fig. 1.1). Promoter lacUV5
initiates the transcription of the downstream genes cI857 and mRFP, leading to a
combined result of red fluorescence and pλ inhibition. While the pλ, just the
contrary,
starts the expression of GFP and lacUV5 repressor LacI. Thus, if inducing the cells
with
IPTG, the inhibition on lacUV5 by LacI will be released, triggering the generation
of
red signal. And the treatment of higher incubation temperature, like 42℃,
promotes
the
degradation of cI protein, inducing the biological function of pλ then causing a
green
signal.

### 3.2 Build

In our project, Golden Gate Assembly (GG) is applied to ligate all the
genes
and their
backbones pET28a+. Thus, a series of DNA fragments with flanking sequences which are
consistent type IIS restriction enzyme recognition sites and complementary
restriction
sites are generated using PCR amplification. Additionally, the 5' overhang of
primers
may also extend the amplicons with some short but fundamental parts like
Shine-Dalgarno
sequences, promoters and terminators.

The figure of agarose gel electrophoresis of DNA fragment that build the
toggle-switch plasmid is shown in Fig. 3.1(a).

![Agarose gel electrophoresis of toggle-switch circuit](https://static.igem.org/mediawiki/2021/3/35/T--XJTU-China--tggl.png)
**Fig. 3.1 Agarose gel electrophoresis of
toggle-switch circuit.** (a) The circuit of toggle switch (3735bp) is
constructed and the electrophoresis result shows the expected band (right lane).
(b) The toggle-switch plasmid has the length of 5920bp. Then the plasmid is
subjected to double
digestion with XbaI and SapI to yield fragments in 3825bp and 2095bp

These two fragments are consequently used for GG to construct the plasmid containing
our
toggle-switch circuit. After transformation to *E.coli* DH5alpha, plasmid
extraction
and a
set of screening including colony PCR and double digestion, strains with expected
plasmid are selected, indicating the toggle-switch circuit is built. And the
sequencing result confirms no mutation in the circuit.

### 3.3 Test

#### 3.3.1 RT-qPCR quantifications of Toggle-Switch

We first used RT-qPCR to detect the transcription of each gene under
different
induction conditions, so as to preliminarily judge the effect of promoter
bistable
expression.

Method

**Method:**
Cultivation: Using LB liquid medium, 37℃, 200rpm. After
cultivating for 3h,
cells
are
cultivated under inducing conditions (1mM IPTG / 42℃) for 8h
respectively.
Total RNA extraction: Using RNAsimple Total RNA Kit,DP419 (TIANGEN
BIOTECH (BEIJING)
CO.,LTD.)
cDNA preparation: Using Evo M-MLV RT Mix (Vazyme Biotech Co.,Ltd);
template
concentration: 50ng RNA/ul; reaction condition: 37℃ 15min,
85℃
15sec.
qPCR: Using ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co.,Ltd).

Relative Normalized Expression data is calculated by using the equation
below,
Relative Expression = 2-[ΔCt(T)-ΔCt(C)]
where ΔCt(T) represents the difference between
Ct value of target gene and internal standard
gene in
treatment
group; ΔCt(C) represents the difference between
Ct value of target gene and internal standard
gene in negative
control group.

**Results and Discussion:** Cells were
induced by two inducers IPTG and 42℃ separately, to test the two state of
toggle
switch under different conditions. As shown in Fig. 3.3, with IPTG induction, the
relative transcriptional level of *mRFP* was significantly increased about 5 folds in
the steady state (a), and *cI857*, that is under the same control of lacUV5 promoter
with *mRFP*, also showed the improved transcriptional level (b), indicating one state
promoted by lacUV5 promoter. In contrast, when cells were incubated in 42℃,
the mRNA
level of *sfGFP* was remarkably enhanced from 1.5x105 to
7.9x106, indicating another
steady state controlled by lamda promoter. The transcription of downstream LacI was
just slightly increased as shown in Fig. 3.3(b), which may attribute to the copy of
lacI
gene in the genome of *E.coli*. In all, induction by 1mM IPTG resulted in obvious
up-regulation of transcription of *mRFP* and *cI857*, while inhibition in *sfGFP* and
LacI. And under 42℃ induction, the relative expression of genes has been
reversed.
The results indicate the two steady states can be achieved under two inducers
respectively.

![RT-qPCR Result](https://static.igem.org/mediawiki/2021/e/e7/T--XJTU-China--engineering3.3.png)
**Fig. 3.3**  Relative normalized expression
of
(a): *mRFP* and *sfGFP* (b): *cI857* and *lacI* in toggle-switch circuit measured by
RT-qPCR

#### 3.3.2 Characterization of Bistability of Toogle-Switch

Method

**Method:**
 Due to the expression of
reporter genes in toggle
switch, GFP
and
RFP
signals are detected by a microplate reader (SpectraMax i3). After
normalization,
that is, dividing the fluorescent signals by corresponding OD600, the intensity of
fluor-signal per cell is gained. And upon inducing with either IPTG or
42℃,
temporal
determination of all the OD600, GFP &RFP
fluorescent
signals provides us a set of
data that could describe the bistable-switch function of our
toggle-switch
circuit.

We used full spectrum scanning to determine the most appropriate
excitation and
emission wavelengths for the two fluorescent proteins. Wavelength used
are as
below:

sfGFP: Excitation 485nm; Emission 535nm
mRFP: Excitation 587nm; Emission 627nm

Cultivation: Using LB liquid medium, 37℃, 200rpm.
Induction: 1mM IPTG was used in 0-719 min culturing. Then the cells were
collected
by centrifugation and replaced with fresh medium without IPTG, and
cultured at
42℃

The obtained fluorescence intensity data was normalized by subtracting
the
intensity
of the negative control (DH5alpha strain containing empty pET8a+ vector)
and
divided
by the 600nm absorbance (representing the thallus concentration).

**Result:**
The bistability of toggle switch was tested by switch of two inducers. As shown in
Fig. 3.4, cells were first induced by IPTG, and *mRFP* was expressed showing stably
increased red fluorescence. Because of the *cI857* was highly expressed in the same
time, lamda promoter was inhibited and little expression of *sfGFP* was observed. At
around 720 min, cells were washed, resuspended in fresh LB medium, and incubated in
42℃. The change of induction condition led to the shifted expression of *sfGFP*
and
LacI, and the expression of *mRFP* was dramatically decreased due to the inhibitory
effect of Lac I on lacUV5 promoter. Fig. 3.4 clearly showed the fast switch of two states
controlled by two inducers, and the bistability can be aslo achieved under each
conditions.

![Characterization Result](https://static.igem.org/mediawiki/2021/1/14/T--XJTU-China--engineering3.4.png)
**Fig. 3.4**  Relative fluorescence
intensity
of *sfGFP* and *mRFP* in toggle-switch circuit under different inducing
conditions

### 3.4 Learn

**Conclusion:** We confirmed the function of toggle-switch circuit in RT-qPCR
and
fluorescence measurement results. We successfully achieved the bistable
expression
of *sfGFP* and *mRFP* under different induction conditions. The alternation of the
two
fluorescence intensities was observed after the change of conditions, reporting
the
intensities of respective promoters.

According to the above results,the feasibility has been proved that we can use
this
circuit to control the expression of *aroG*, *trpBA* and *pykA* genes, so as to
control
the cell into different states of “proliferation” and “production”.

---

Read more results of our new parts at [Proof of Concept](https://2021.igem.org/Team%3AXJTU-China/Proof_Of_Concept) or [improvement](https://2021.igem.org/Team%3AXJTU-China/Improve).

For protocols we apply in our labwork, see also: [Protocols](https://2021.igem.org/Team%3AXJTU-China/Protocols)

MENU

* [home](/Team%3AXJTU-China)
* [Project](/Team%3AXJTU-China/Description)
  + [description](/Team%3AXJTU-China/Description)
  + [design](/Team%3AXJTU-China/Design)
  + [implementation](/Team%3AXJTU-China/Implementation)
  + [parts](/Team%3AXJTU-China/Parts)
* wetlab
  + [engineering](/Team%3AXJTU-China/Engineering)
  + [improvement](/Team%3AXJTU-China/Improve)
  + [Proof
    of Concept](/Team%3AXJTU-China/Proof_Of_Concept)
  + [protocols](/Team%3AXJTU-China/Protocols)
* drylab
  + [modelling](/Team%3AXJTU-China/Model)
  + [protein modelling](/Team%3AXJTU-China/protein_model)
  + [hardware](/Team%3AXJTU-China/Hardware)
* Human Practices
  + [overview](/Team%3AXJTU-China/Human_Practices)
  + [interview](/Team%3AXJTU-China/Human_Practices#inspiration)
  + [visit](/Team%3AXJTU-China/Human_Practices#point_6)
  + [survey](/Team%3AXJTU-China/Human_Practices#point_7)
  + [Communication](/Team%3AXJTU-China/Communication)
  + [Education](/Team%3AXJTU-China/Education)
* Team
  + [crew](/Team%3AXJTU-China/Team)
  + [attributions](/Team%3AXJTU-China/Attributions)
  + [contribution](/Team%3AXJTU-China/Contribution)
  + [collaboration](/Team%3AXJTU-China/Collaborations)
  + [partnership](/Team%3AXJTU-China/Partnership)

#### contact us

**Xi'an Jiaotong University**
28 Xianning West Road
Xi'an, Shaanxi, China, 710049

xjtu\_igem@xjtu.edu.cn

#### follow us

---

#### special thanks to

[![Xi'an Jiaotong University](https://static.igem.org/mediawiki/2021/1/15/T--XJTU-China--XJTU.png)](http://www.xjtu.edu.cn/)

[![School of Life Science and Technology](https://static.igem.org/mediawiki/2021/9/9f/T--XJTU-China--slst-logo.png)](http://slst.xjtu.edu.cn/)

[![Qian Xuesen Honor College](https://static.igem.org/mediawiki/2021/b/b3/T--XJTU-China--Qian_Xuesen_College.png)](http://bjb.xjtu.edu.cn/)
![Ailurus](https://static.igem.org/mediawiki/2021/4/48/T--XJTU-China--Ailurus.png)
[![Bluepha](https://static.igem.org/mediawiki/2021/8/8e/T--XJTU-China--Bluepha.png)](http://www.bluepha.com/)

Made with ❤️ by

[XJTU-iGEMers](/Team%3AXJTU-China/Team#XJTU-iGEMers)

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

# Team:XJTU-China/Experiments

1. sideMenu, #top\_title, .patrollink, #firstHeading, #home\_logo, #sideMenu,a#top,p:empty, #top\_menu\_under { display:none; }
2. content { padding:0px; width:100%; margin-top:-7px; margin-left:0px; border:none;}
3. bodyContent h1, #bodyContent h2, #bodyContent h3, #bodyContent h4, #bodyContent h5 { margin-bottom: 0px; }
4. bodyContent a[href ^="https://"], .link-https { padding-right:0px;}
5. globalWrapper {padding-bottom:0}

# Experiments

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

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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

# Team:XJTU-China/Results

1. sideMenu, #top\_title, .patrollink, #firstHeading, #home\_logo, #sideMenu,a#top,p:empty, #top\_menu\_under { display:none; }
2. content { padding:0px; width:100%; margin-top:-7px; margin-left:0px; border:none;}
3. bodyContent h1, #bodyContent h2, #bodyContent h3, #bodyContent h4, #bodyContent h5 { margin-bottom: 0px; }
4. bodyContent a[href ^="https://"], .link-https { padding-right:0px;}
5. globalWrapper {padding-bottom:0}

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

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

### Describe what your results mean

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

### Project Achievements

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

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

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)