iGEM 2020 — Team NJMU-China

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Model

Notebook

### Construction of 5-HT Biosensor

Construction Time 1(from synthesis company)

Construction Time 2(from synthesis company)

July

August

September

October

#### Construction Time 1

#### Construction Time 2

#### July

#### August

#### September

#### October

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# Team:NJMU-China/Engineering

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## Overview

We have successfully completed the synthesis of our parts. Also, we simulated the interactions and verified success of our engineering preliminarily.

## Success in Constructing Parts

In the first place, we have successfully optimized the color-reaction reporting system (Figure 1).

![](https://static.igem.org/mediawiki/2020/c/ca/T--NJMU-China--eng.png)

Figure 1. Color-Reaction of pGEX-6P-1

Then, we have successfully constructed all the parts and sequenced them for verification.

![](https://static.igem.org/mediawiki/2020/6/68/T--NJMU-China--Enginneering02.png)

Figure 2. Enzyme Digestion Result of Serotonin Biosensor Version 2

## Demonstration of Feasibility about Our Design

We successfully constructed the 3D structure of proteins (LasR, LacZ), DNA fragments (PlasI) and small molecules (Serotonin, Dopamine, 3-O-C12-HSL, X-gal) involved in this project (See more in [Simulations of Interaction](https://2020.igem.org/Team%3ANJMU-China/Simulation_of_Interactions) ).

We evaluated all interactions involved in our project by calculating docking scores. The results show that, the docking of LasR-Serotonin/Dopamine ~ PlasI have scores of -260.09 and -258.87 (the greater the absolute value, the more informative) (Table 1). We also computed the docking score of LasR-HSL ~ PlasI (Positive Control) and LasR ~ PlasI (Negative Control). We found that the docking of LasR-Serotonin/Dopamine ~ PlasI have almost same score with the docking of LasR-HSL ~ PlasI at similar ligand RMSD, while having apparently higher score in comparison with the docking of LasR ~ PlasI.

In summary, the protein complex of LasR-Serotonin has similar performance with the experimental verified LasR-HSL complex in docking with PlasI, indicating its feasibility to be designed into a biosensor.

|  |  |  |
| --- | --- | --- |
| Interaction Type | Docking score | Ligand rmsd (Å) |
| LasR-HSL/PlasI | -257.85 | 135.92 |
| LasR-serotonin/PlasI | -260.09 | 135.53 |
| LasR-dopamine/PlasI | -258.87 | 167.30 |
| LasR/PlasI | -229.76 | 174.41 |

Table 1. Docking Scores of Different Interactions

##

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---

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# Team:NJMU-China/Experiments

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# Experiments

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# Team:NJMU-China/Results

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![](https://static.igem.org/mediawiki/2020/b/b2/T--NJMU-China--Four.png)
[![](https://static.igem.org/mediawiki/2020/3/38/T--NJMU-China--FourOne.png)
[Overview](#stepOne)](#stepOne)
[![](https://static.igem.org/mediawiki/2020/1/12/T--NJMU-China--FourTwo.png)
[Design of Biosensors](#stepTwo)](#stepTwo)
[![](https://static.igem.org/mediawiki/2020/e/e5/T--NJMU-China--FourThree.png)
[Synthesis and Modification](#stepThree)](#stepThree)
[![](https://static.igem.org/mediawiki/2020/2/22/T--NJMU-China--FourFour.png)
[References](#stepFour)](#stepFour)
[![]()](#stepFive)

## Overview

As for the lab work, we have completed the design and synthesis of plasmids as well as the optimization
of the color-reaction reporting system. However, held by the pandemic of COVID-19, we didn’t have access
to lab works until August. Foreseen this situation, we have initiated our modeling project to simulate
the whole interaction as a preliminary estimation of part function (See More in Our MODEL).

## Design of Serotonin Biosensors

There exist no designed biosensors against serotonin. Therefore, we decided to design the serotonin
biosensor from scratch. Through massive literature investigation, we found that in a novel mouse
infection model 1, serotonin, acting as an interboundary signaling molecule through quorum sensing,
stimulates the production of bacterial virulence factors and increases the formation of biofilms.
Specifically, no response was noted in the rhl cellular system, indicating specificity for the las QS
pathway.

![](https://static.igem.org/mediawiki/2020/3/3d/T--NJMU-China--Design3.png)

Based on this knowledge, we referenced the design of biosensors aimed to detect QS molecules 2. The
biosensor plasmid is based on a typical heterologous expression system, pGEX-6p-1 vector and contains
two parts. The first part (Sensor-1) expresses LasR molecule under the control of Ptac promoter and lac
operator (activated in the presence of IPTG). The second part (Sensor-2 & Reporter) is under the control
of the inducible promoter PlasI and expresses beta-galactosidase, which is a classic reporter gene. In
the presence of serotonin or Quorum-Sensing molecules (e.g. HSL or AHL), they will bind with LasR to
form a transcription activating complex targeting PlasI. We aimed to visualize the result through
color-reactions by beta-galactosidase and X-gal.

![](https://static.igem.org/mediawiki/2020/9/94/T--NJMU-China--Result2.png)

## Synthesis and Modification of Plasmids

### 1. Synthesis of Sensor-1

![](https://static.igem.org/mediawiki/2020/4/4f/T--NJMU-China--Result3.png)

The gene sequences corresponding to lasR from the lasR/lasI AHL-mediated QS regulatory system of
Pseudomonas aeruginosa were synthesized using polymerase chain reaction (PCR)
(https://www.ncbi.nlm.nih.gov/gene/881789). The plasmid pGEX-6p-1 was prepared by inserting a 5'
BamHI/EcoRI, 3' EcoRI fragment containing the lasR sequences at the downstream of GST and HIV 3C site
(Figure 1). The resulting pGEX-6p-1 vector (Serotonin Biosensor Version 0) contains the lasR gene which
is under the transcriptional control of Ptac.

![](https://static.igem.org/mediawiki/2020/4/45/T--NJMU-China--Result4.png)

Figure 1. Selection of Restriction Endonuclease Sites

The construction of Serotonin Biosensor Version 0 was verified by digesting the plasmid with BamHI and
EcoRI and confirming the lengths of the digested fragments on a 1% agarose gel in 37°C water-bath for 40
minutes (Figure 2). We have also sequenced the engineered part to ensure there are no mutations.

![](https://static.igem.org/mediawiki/2020/a/ae/T--NJMU-China--Result5.png)

Figure 2. Enzyme Digestion Result of Serotonin Biosensor Version 0

### 2. Synthesis of Sensor-2 & Reporter

![](https://static.igem.org/mediawiki/2020/5/5a/T--NJMU-China--Result6.png)

The gene sequences corresponding to PlasI were obtained from the paper 'The quorum-sensing negative
regulator RsaL of Pseudomonas aeruginosa binds to the lasI promoter' published in J Bacteriol 3 (Figure
3). Sequences of related protein ORF were acquired from NCBI: https://www.ncbi.nlm.nih.gov/gene/881777.
We further used homologous recombination methods to integrate this DNA fragment into Serotonin Biosensor
Version 0. The resulting plasmid (Serotonin Biosensor Version 1) contains the lacZ gene which is under
the transcriptional control of PlasI.

![](https://static.igem.org/mediawiki/2020/1/19/T--NJMU-China--Result7.png)

The construction of Serotonin Biosensor Version 1 was verified by digesting the plasmid with Clal and
Xhol and confirming the lengths of the digested fragments on a 1% agarose gel in 37°C water-bath for 40
minutes (Figure 4). We have also sequenced the engineered part to ensure there are no mutations.

![](https://static.igem.org/mediawiki/2020/b/b1/T--NJMU-China--Result8.png)

Figure 4. Enzyme Digestion Result of Serotonin Biosensor Version 1

### 3. Optimization of the color-reaction reporting system

Before characterization of our constructed parts, we have optimized the color-reaction reporting system
as we have introduced other reagents like standard serotonin in the system. Through gradient-condition
experiments using wild-type pGEX-6p-1 vectors，we have found the optimal amount of IPTG and X-gal as
shown below:

## References

1 Knecht, L. D. et al. Serotonin Activates Bacterial Quorum Sensing and Enhances the Virulence
of Pseudomonas aeruginosa in the Host. EBioMedicine 9, 161-169, doi:10.1016/j.ebiom.2016.05.037
(2016).
2 Saeidi, N. et al. Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human
pathogen. Mol Syst Biol 7, 521, doi:10.1038/msb.2011.55 (2011).
3 Rampioni, G. et al. The quorum-sensing negative regulator RsaL of Pseudomonas aeruginosa binds to the
lasI promoter. J Bacteriol 188, 815-819, doi:10.1128/JB.188.2.815-819.2006 (2006).
4 Struss, A., Pasini, P., Ensor, C. M., Raut, N. & Daunert, S. Paper strip whole cell biosensors: a
portable test for the semiquantitative detection of bacterial quorum signaling molecules. Anal Chem 82,
4457-4463, doi:10.1021/ac100231a (2010).
5 Jayaprada, T. et al. The interference of nonylphenol with bacterial cell-to-cell communication.
Environ Pollut 257, 113352, doi:10.1016/j.envpol.2019.113352 (2020).

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---

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