iGEM 2019 — Team RDFZ-China

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

# Team:RDFZ-China/Notebook

* [Home](https://2019.igem.org/Team%3ARDFZ-China "Home")
* [Project](https://2019.igem.org/Team%3ARDFZ-China/Project_Description)

  [Description](https://2019.igem.org/Team%3ARDFZ-China/Project_Description)
  [Design](https://2019.igem.org/Team%3ARDFZ-China/Design)
  [Demonstrate](https://2019.igem.org/Team%3ARDFZ-China/Demonstrate)
  [Search Engine](https://2019.igem.org/Team%3ARDFZ-China/Search_Engine)
* [Model](https://2019.igem.org/Team%3ARDFZ-China/Model)
* [Human Practice](https://2019.igem.org/Team%3ARDFZ-China/Result)

  [Integrated](https://2019.igem.org/Team%3ARDFZ-China/Human_Practices)
  [Public Engagement & Education](https://2019.igem.org/Team%3ARDFZ-China/Engagement)
  [Future Plan](https://2019.igem.org/Team%3ARDFZ-China/Measurement)
* [Part](https://2019.igem.org/Team%3ARDFZ-China/Basic_xPart)

  [Part collection](https://2019.igem.org/Team%3ARDFZ-China/Basic_xPart)
  [Basic Parts](https://2019.igem.org/Team%3ARDFZ-China/Collection)
* [Safety](https://2019.igem.org/Team%3ARDFZ-China/Safety "Safety")
* [Laboratory](https://2019.igem.org/Team%3ARDFZ-China/Result)

  [Result](https://2019.igem.org/Team%3ARDFZ-China/Result)
  [Protocol](https://static.igem.org/mediawiki/2019/7/71/T--RDFZ-China--Public.pdf)
  [Notebook](https://static.igem.org/mediawiki/2019/7/72/T--RDFZ-China--notes.pdf)
* [Team](https://2019.igem.org/Team%3ARDFZ-China/Members)

  [Collaboration](https://2019.igem.org/Team%3ARDFZ-China/Collaborations_1)
  [Members](https://2019.igem.org/Team%3ARDFZ-China/Members)
  [Attribution & Acknowledgement](https://2019.igem.org/Team%3ARDFZ-China/Attributions)
  [School](https://2019.igem.org/Team%3ARDFZ-China/School)
* [Achievement](https://2019.igem.org/Team%3ARDFZ-China/Achievement "Achievement")

# 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)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

* + <a href="#section1">Bound Fraction & Unbound Fraction</a>
  + <a href="#section2">Modelling Equations</a>
  + <a href="#section3">Methods&Results</a>
  + <a href="#section4">Discussion</a>

## Contents

* [1 Modelling](#Modelling)
  + [1.1 Bound Fraction & Unbound Fraction](#Bound_Fraction_.26_Unbound_Fraction)
  + [1.2 Modelling Equations](#Modelling_Equations)
  + [1.3 Methods&Results](#Methods.26Results)
  + [1.4 Discussion](#Discussion)
  + [1.5 References](#References)

# Modelling

### Bound Fraction & Unbound Fraction

By defining a simple equation where P is the promoter region and X is the ligand that
can bind with P, which is an activator or a repressor:

<img src="![T--RDFZ-China--reaction.jpeg](https://static.igem.org/mediawiki/2018/c/c4/T--RDFZ-China--reaction.jpeg)

The factional saturation of the promoter is denoted by Ps , which is the fraction of
occupied binding sites:

<img src="![T--RDFZ-China--equation11.jpeg](https://static.igem.org/mediawiki/2018/3/3d/T--RDFZ-China--equation11.jpeg)"/style="text-align:center;">

At equilibrium of this reaction, the rate of forward reaction and that of backward reaction are
the same:

<img src="![T--RDFZ-China--equation12.jpeg](https://static.igem.org/mediawiki/2018/a/ad/T--RDFZ-China--equation12.jpeg)">

Then we rearrange this equation and get:

<img src="![T--rdfZ-China--equation13.jpeg](https://static.igem.org/mediawiki/2018/6/63/T--rdfZ-China--equation13.jpeg)"/>

Where kd is the dissociation constant of this binding event. Now we substitute equation 1.3 into equation 1.1 and hence obtain:

<img src="![T--rdfZ-China--equation14.jpeg](https://static.igem.org/mediawiki/2018/7/77/T--rdfZ-China--equation14.jpeg)"/>

Assuming that the sum of the bound and the unbound equals one, the unbound fraction Pu is thus:

<img src="![T--rdfZ-China--equation15.jpeg](https://static.igem.org/mediawiki/2018/f/f2/T--rdfZ-China--equation15.jpeg)"/>

Bound fraction is considered for activation in gene regulation and unbound fraction for repressive regulation.

### Modelling Equations

<img src="![T--RDFZ-China--Equation_1.png](https://static.igem.org/mediawiki/2018/e/ec/T--RDFZ-China--Equation_1.png)"/>

The chemical AHL (A) is required by LuxR to activate transcription. The active form for LuxR it consists of a dimer of the
complex LuxR-AHL ((Y:A)2). Thus we can consider the production of this dimer as an elementary reaction: 2A+2Y --> (Y:A)2,
whose rate depends upon A, the concentration of AHL, and the concentration of unbound LuxR monomer, Runbound.

<img src="![T--RDFZ-China--Equation_2.png](https://static.igem.org/mediawiki/2018/a/a4/T--RDFZ-China--Equation_2.png)"/>

Where synthesis is the rate of synthesis of the dimer, k1 is the binding coefficient RT is the total concentration of LuxR
monomer, and R\*(t) is the concentration of AHL-LuxR dimer.
And k2R\*(t) is simply the rate of degradation of the dimer, where k2 is the degradation coefficient of AHL-LuxR.

<img src="![T--RDFZ-China--Equation_3.png](https://static.igem.org/mediawiki/2018/b/bc/T--RDFZ-China--Equation_3.png)"/>

For any given mRNA:

<img src="![T--RDFZ-China--Equation_4.png](https://static.igem.org/mediawiki/2018/a/ad/T--RDFZ-China--Equation_4.png)">

Where m(t) is the concentration of mRNA, k0 is the amount of mRNA produced, δm is the degradation rate of mRNA.
For PhIF protein translated from the given mRNA:

<img src="![T--RDFZ-China--Equation_5.png](https://static.igem.org/mediawiki/2018/2/20/T--RDFZ-China--Equation_5.png)"/>

Where P(t) is the concentration of PhIF protein, k3 is the translation rate, and δp is the degradation rate of PhIF
protein.
Then substitute equation 1.4 into the calculation of k0 (for the activation of Plux promoter by AHL-LuxR correspond to
bound fraction):

<img src="![T--RDFZ-China--Equation_6.png](https://static.igem.org/mediawiki/2018/d/db/T--RDFZ-China--Equation_6.png)"/>

Where a is the transcription rate, k2 is the equivalent of kd in equation 1.4 and 1.5.

<img src="![T--RDFZ-China--Equation_7.png](https://static.igem.org/mediawiki/2018/3/32/T--RDFZ-China--Equation_7.png)"/>

For sfGFP protein translated from the given mRNA:

<img src="![T--RDFZ-China--Equation_8.png](https://static.igem.org/mediawiki/2018/5/50/T--RDFZ-China--Equation_8.png)"/>

Where G(t) is the concentration of sfGFP protein, k3 is the translation rate, and δg is the degradation rate of PhIF
protein.
Then substitute equation 1.5 into the calculation of k0 in this case (for the repression of PPhIF promoter by PhIF protein
correspond to the unbound fraction):

<img src="![T--RDFZ-China--Equation_9.png](https://static.igem.org/mediawiki/2018/2/24/T--RDFZ-China--Equation_9.png)"/>

Where a is the transcription rate, k2 is the equivalent of kd in equation 1.4 and 1.5.

### Methods&Results

To simulate our model, we use the Matlab ODE suite to solve three differential equations simultaneously. A breif diagram of the regulatory network can be found below:

<img src="![T--RDFZ-China--quorum_sensing_model.jpeg](https://static.igem.org/mediawiki/2018/1/1e/T--RDFZ-China--quorum_sensing_model.jpeg)"/>

By adjusting the initial dose of AHL, ranging from 10^-5 M to 10^-12 M , we can get a variance of dose response below:

<img src="![T--RDFZ-China--parameterScanGif.gif](https://static.igem.org/mediawiki/2018/a/a2/T--RDFZ-China--parameterScanGif.gif)"/>

From the simulation results, we can see that between [AHL]=10^-10 and 10^-11 M, the concentration of the sfGFP(which represents ccdb) rises above 5\*10^-8M which gives the half maximal killing rate, meaning a large proportion of bacteria in the fermentor would die. Therefore, the worker in the factory should always be aware not having [AHL] below that range.

<img src="![T--RDFZ-China--AHL_parameter_scan.jpeg](https://static.igem.org/mediawiki/2018/1/17/T--RDFZ-China--AHL_parameter_scan.jpeg)"/width="80%;">

A local sensitivity analysis is also carried out to help us increase the robustness of the model. Two influential factors are selected to perform a further parameter scan.

<img src="![T--RDFZ-China--sensitivity_matrix.jpeg](https://static.igem.org/mediawiki/2018/9/9a/T--RDFZ-China--sensitivity_matrix.jpeg)"/width="80%;">

<img src="![T--RDFZ-China--sensitivity_figure.jpeg](https://static.igem.org/mediawiki/2018/1/14/T--RDFZ-China--sensitivity_figure.jpeg)"/width="80%;">

<img src="![T--RDFZ-China--scanning_figure_K1.jpeg](https://static.igem.org/mediawiki/2018/6/6d/T--RDFZ-China--scanning_figure_K1.jpeg)"/width="50%;"><img src="![T--RDFZ-China--scanning_figure_K2.jpeg](https://static.igem.org/mediawiki/2018/8/83/T--RDFZ-China--scanning_figure_K2.jpeg)"/width="50%;">

The scanning results suggest that the variation of the parameter, K1 and K2, by 10% respectively will not affect the trend.

Furthermore, our results from the wetlab indicates a similar trend, corroborating our results from modelling

<img src="![T--RDFZ-China--Lux-characterization.png](https://static.igem.org/mediawiki/2018/2/28/T--RDFZ-China--Lux-characterization.png)"/width="80%;">

### Discussion

In a real factory, there will be two fermentors, one for the production of AHL which helps keeping the bacteria in the second fermentor alive, one for the production of the main product. Theoretically, by measuring the OD in the first fermentor, the concentration of AHL can be inferred by using an algorithm shown below.

<img src="![T--RDFZ-China--concept_proof.png](https://static.igem.org/mediawiki/2018/4/4a/T--RDFZ-China--concept_proof.png)"/width="80%;">

<img src="![T--RDFZ-China--proof_concept_label.png](https://static.igem.org/mediawiki/2018/8/8c/T--RDFZ-China--proof_concept_label.png)"/width="80%;">

In conclusion, our model sheds light on the amount of concentration of AHL in the fermentor that is necessary for keeping the bacteria alive. The model simulation results correspond well to the data obtained from the wet-lab experiment. In addition, we also attempt to derive an algorithm to help measure the AHL concentration in the other fermentor.

### References

[1] Basu, S., Mehreja, R., Thiberge, S., Chen, M., & Weiss, R. (2004). Spatiotemporal control of gene expression with pulse-generating networks. Proceedings Of The National Academy Of Sciences, 101(17), 6355-6360. doi:10.1073/pnas.0307571101

[2] Ingalls, Brian P. Mathematical Modeling in Systems Biology: an Introduction. MIT Press, 2013.

[3]"Luxi - Acyl-Homoserine-Lactone Synthase - Aliivibrio Fischeri - Luxi Gene & Protein". 2018. Uniprot.Org. Accessed October 18 2018. <https://www.uniprot.org/uniprot/P12747>.

[4]Rodrigo, G., Carrera, J., Elena, S. F., & Jaramillo, A. (2010). Robust dynamical pattern formation from a multifunctional minimal genetic circuit. BMC systems biology, 4(1), 48.

[5] Smith, R., Tan, C., Srimani, J., Pai, A., Riccione, K., Song, H., & You, L. (2014). Programmed Allee effect in bacteria causes a tradeoff between population spread and survival. Proceedings Of The National Academy Of Sciences, 111(5), 1969-1974. doi:10.1073/pnas.1315954111

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

# Team:RDFZ-China/Experiments

* [Home](https://2019.igem.org/Team%3ARDFZ-China "Home")
* [Project](https://2019.igem.org/Team%3ARDFZ-China/Project_Description)

  [Description](https://2019.igem.org/Team%3ARDFZ-China/Project_Description)
  [Design](https://2019.igem.org/Team%3ARDFZ-China/Design)
  [Demonstrate](https://2019.igem.org/Team%3ARDFZ-China/Demonstrate)
  [Search Engine](https://2019.igem.org/Team%3ARDFZ-China/Search_Engine)
* [Model](https://2019.igem.org/Team%3ARDFZ-China/Model)
* [Human Practice](https://2019.igem.org/Team%3ARDFZ-China/Result)

  [Integrated](https://2019.igem.org/Team%3ARDFZ-China/Human_Practices)
  [Public Engagement & Education](https://2019.igem.org/Team%3ARDFZ-China/Engagement)
  [Future Plan](https://2019.igem.org/Team%3ARDFZ-China/Measurement)
* [Part](https://2019.igem.org/Team%3ARDFZ-China/Basic_xPart)

  [Part collection](https://2019.igem.org/Team%3ARDFZ-China/Basic_xPart)
  [Basic Parts](https://2019.igem.org/Team%3ARDFZ-China/Collection)
* [Safety](https://2019.igem.org/Team%3ARDFZ-China/Safety "Safety")
* [Laboratory](https://2019.igem.org/Team%3ARDFZ-China/Result)

  [Result](https://2019.igem.org/Team%3ARDFZ-China/Result)
  [Protocol](https://static.igem.org/mediawiki/2019/7/71/T--RDFZ-China--Public.pdf)
  [Notebook](https://static.igem.org/mediawiki/2019/7/72/T--RDFZ-China--notes.pdf)
* [Team](https://2019.igem.org/Team%3ARDFZ-China/Members)

  [Collaboration](https://2019.igem.org/Team%3ARDFZ-China/Collaborations_1)
  [Members](https://2019.igem.org/Team%3ARDFZ-China/Members)
  [Attribution & Acknowledgement](https://2019.igem.org/Team%3ARDFZ-China/Attributions)
  [School](https://2019.igem.org/Team%3ARDFZ-China/School)
* [Achievement](https://2019.igem.org/Team%3ARDFZ-China/Achievement "Achievement")

# 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.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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

# Team:RDFZ-China/Results

* [Home](https://2019.igem.org/Team%3ARDFZ-China "Home")
* [Project](https://2019.igem.org/Team%3ARDFZ-China/Project_Description)

  [Description](https://2019.igem.org/Team%3ARDFZ-China/Project_Description)
  [Design](https://2019.igem.org/Team%3ARDFZ-China/Design)
  [Demonstrate](https://2019.igem.org/Team%3ARDFZ-China/Demonstrate)
  [Search Engine](https://2019.igem.org/Team%3ARDFZ-China/Search_Engine)
* [Model](https://2019.igem.org/Team%3ARDFZ-China/Model)
* [Human Practice](https://2019.igem.org/Team%3ARDFZ-China/Result)

  [Integrated](https://2019.igem.org/Team%3ARDFZ-China/Human_Practices)
  [Public Engagement & Education](https://2019.igem.org/Team%3ARDFZ-China/Engagement)
  [Future Plan](https://2019.igem.org/Team%3ARDFZ-China/Measurement)
* [Part](https://2019.igem.org/Team%3ARDFZ-China/Basic_xPart)

  [Part collection](https://2019.igem.org/Team%3ARDFZ-China/Basic_xPart)
  [Basic Parts](https://2019.igem.org/Team%3ARDFZ-China/Collection)
* [Safety](https://2019.igem.org/Team%3ARDFZ-China/Safety "Safety")
* [Laboratory](https://2019.igem.org/Team%3ARDFZ-China/Result)

  [Result](https://2019.igem.org/Team%3ARDFZ-China/Result)
  [Protocol](https://static.igem.org/mediawiki/2019/7/71/T--RDFZ-China--Public.pdf)
  [Notebook](https://static.igem.org/mediawiki/2019/7/72/T--RDFZ-China--notes.pdf)
* [Team](https://2019.igem.org/Team%3ARDFZ-China/Members)

  [Collaboration](https://2019.igem.org/Team%3ARDFZ-China/Collaborations_1)
  [Members](https://2019.igem.org/Team%3ARDFZ-China/Members)
  [Attribution & Acknowledgement](https://2019.igem.org/Team%3ARDFZ-China/Attributions)
  [School](https://2019.igem.org/Team%3ARDFZ-China/School)
* [Achievement](https://2019.igem.org/Team%3ARDFZ-China/Achievement "Achievement")

# Results

Here you can describe the results of your project and your future plans.

### 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.** 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.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)

​