iGEM 2023 — Team hkgtc

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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Content

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[* 2022](#PI)[* 2023](#HOW)

ㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤㅤ

February 2022

11/2

Brainstorm meeting

25/2

Brainstorm meeting

March 2022

21/3

Brainstorm meeting

30/3

Brainstorm meeting

May 2022

2/5

Brainstorm meeting

June 2022

17/6

Brainstorm meeting

Aug 2022

20/8

Recruit team member

September 2022

13/9

Confirmation of 2023 HKGTC iGEM topic

October 2022 - January 2023

Research proposal preparation

February 2023

3/2

Learning of Hill function

7/2

Data Science Experience Course-Plastic tagging activity

March 2023

20/3

First construct design

23/3

iGEM sharing session

April 2023

7/4

Beach-cleanup pre-trip

20/4

Interview with Dr. Wright

26/4

Data Science Experience Course-Plastic tagging activity

May 2023

10/5

iGEM sharing session

13/5

Designed the first prototype of Microplastics Data Collection Car

17/5

Interview with Leanne Tam from Greenpeace

20/5

Beach clean-up activity

23/5

AHL induction with construct #1 and #3

25/5

Seek advice from school teacher, Mr Alex O

Second design

27/5

Proposed 2nd and 3rd phase of machine making

30/5

AHL induction with construct #1 and #3

Interview with Prof. Patrick Lee from the City University of Hong Kong

31/5

Full proposal of machine made

June 2023

3/6

Proposed ‘making 3D graphs for microplastics distribution’ in phase 3

6/6

AHL induction with construct #1 and #3

7/6

Survey

11/6

Interview with Oxford University iGEM team

14/6

Survey, interview with Harvard University iGEM team

13/6

AHL induction with construct #1 and #3

14/6

AHL induction with construct #1 and #3

30/6

Rehearsal for green fashion show

July 2023

1/7

Starts to study the performance of biosensors

3/7

Green fashion show, interview with Prof. King Chow from The Hong Kong University of Science and Technology

4/7

Recruited Clifford Wong to assist the building and design of the machine

Interview with EPD

5/7

New design of the machine by Clifford

iGEM sharing session

6/7

Interview with Environmental Protection Department (EPD)

11/7

Collaboration meeting with Oxford

13/7

AHL induction with construct #1 and #4

14/7

Collaboration meeting with Harvard

15/7

Building the machine and making 3d printing models

19/7

AHL induction with construct #1, #3, and #4

20/7

AHL induction with construct #2, #1 2:1, and #1 3:1

21/7

AHL induction with construct #2, #1 1:1, #1 2:1, and #1 3:1

23/7

Meeting with Jonas Fok, advisor

28/7

AHL induction but with all negative samples

August 2023

1/8

AHL induction with construct #1, #2, #3, and #4 with addition of IPTG

2/8

AHL induction with construct #1 1:1, #1 2:1, and #1 3:1

AHL induction with construct #1 3:1

Collaboration meeting with Oxford

3/8

AHL induction with construct #1 1:1 and #1 3:1

September 2023

2/9

New design of AI car

11/9

Project exhibition

23/9

AHL induction with construct pSB1C3-LasR-pLasRL-EGFP

New design of AI car

Project logo design confirmed

26/9

AHL induction with 3 selected colonies of construct pSB1C3-LasR-pLasRL-EGFP, starting experiments with triplicate results

28/9

AHL induction with 1 selected colony of construct pSB1C3-LasR-pLasRL-EGFP with different types of AHL

30/9

AHL induction with 1 selected colony of construct pSB1C3-LasR-pLasRL-EGFP with AHL 3OC12-HSL and different AHL concentrations

October 2023

1/10

AHL induction with 1 selected colony of construct pSB1C3-LasR-pLasRL-EGFP with AHL 3OC12-HSL and different AHL concentrations

3/10

Project exhibition

5/10

Project exhibition

6/10

Project exhibition

7 - 12/10

Wiki content preparation

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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Content

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[* Summary](#summary)[* (A) OD600 and EGFP production of the three different ratios of a biosensor](#A)[* (B) Fluorescence images of the three different ratios of a biosensor](#B)[* (C) OD600 and EGFP production of the four biosensors incubated with a cocktail of synthetic AHL molecules](#C)[* (D) Fluorescence images of the four biosensors incubated with a cocktail of synthetic AHL molecules](#D)[* (E) OD600 and EGFP production of pSB1C3-LasR-pLasRL-EGFP incubated with a cocktail of synthetic AHL molecules](#E)[* (F) Fluorescence images of pSB1C3-LasR-pLasRL-EGFP incubated with a cocktail of synthetic AHL molecules](#F)[* Future perspectives](#Fp)

Summary

Microplastics has been identified as hubs for microbial pathogens and their antimicrobial resistance (AMR) due to hydrophobicity, greater surface area and pit forming nature of microplastics. These pathogenic bacteria form biofilms on the microplastics. Biofilm formation is governed by the quorum sensing (QS) genes expressed as a result of the accumulation of a critical cell density. *Pseudomonas* species has been reported to have the abilities of attaching to the microplastics and are contributing to microplastic-associated AMR. It employs N-acyl homoserine lactone (AHL) as their command language to coordinate population behaviour during invasion and colonization. There is a complete quorum sensing regulatory circuit LasI-LasR in *P. aeruginosa* . In our project, we engineered a whole cell biosensor based on the LasI-LasR system to detect and quantitatively measure the presence of quorum sensing molecules secreted from *P. aeruginosa* for detection of microplastic pollution levels in water samples. We went through a cycle of engineering (Research → Design → Build → Test → Learn → Improve).

Research

From literature reviews, we found that the engineering of whole-cell biosensors based on quorum sensing systems is mainly for biomedical application while the potential of biosensors for detection of microplastic level is unexplored. Thus we aimed to apply *P. aeruginosa* LasI-LasR circuit to engineer the proof-of-concept biosensor for the detection of specific AHL molecules. The biosensor will ultimately use to quantitatively measure the presence of AHL molecules in water samples for monitoring microplastic pollution.

Design

The engineered biosensor is composed of two modules: the sensing module and reporting module. The sensing module expresses the transcription factor LasR which can bind to the AHL molecule that are secreted from *P. aeruginosa*. Then the formation of LasR-AHL complex can bind to an inducible promoter, which lead to the activation of the reporting module. A green fluorescent protein-based system is used as the reporting module, in which the expression of GFP would be induced by the LasR-AHL complex. The highly efficient expression and easy detection of the green fluorescence signal could facilitate the characterization and optimization of the biosensor system.

Build

The transcription factor LasR was expressed under the control of the constitutive T7 promoter in the plasmid pET 21b and pET 23b. pLasR3 and pLasRL promoters were used as inducible promoters to control the expression of EGFP in pUC57 plasmid. The plasmids pET21b-LasR/ pET23b-LasR and pUC57-pLasRL-EGFP/pUC57-pLasR3-EGFP were transformed into the BL21 C41(DE3)pLysS. The resulting biosensors were named as pET21b-LasR-pUC57-pLasRL-EGFP, pET21b-LasR-pUC57-pLasR3-EGFP, pET23b-LasR-pUC57-pLasRL-EGFP and pET23b-LasR-pUC57-pLasR3-EGFP. An AHL cocktails containing three synthetic AHL molecules, N-butyryl L-homoserine lactone (C4HSL), N-3-oxo-decanoyl L-homoserine lactone (C10HSL) and N-3-oxo-dodecanoyl L-homoserine lactone (3OC12-HSL) were used to determine the specificity and sensitivity of the engineered biosensor.

Test, learn and improve

Several induction tests of the four biosensors with AHL cocktails at published relevant concentrations in water environments from 1x10-5 to 1x10-10 were performed. The EGFP production rates were low as indicated by both weak fluorescence images shown by the fluorescence microscope and fluorescence intensity measured by the microplate reader readings. Stronger fluorescence signals were obtained after several modifications in AHL induction protocol. In the process, we learned that OD600 of 0.6 in starter cell culture was better than 0.3 as proposed by published protocol as the bacteria were at exponential phase when OD600 reached 0.6. Besides, we found that the presence of dimethyl sulfoxide (DMSO) used to make AHL stock solutions and subsequent dilutions caused high autofluorescence signal. Thus, DMSO was used to make AHL stock solutions and distilled water was used in making the subsequent serial dilutions of AHL solutions. To find out if the presence of more pET21b-LasR would allow the formation of more LasR-AHL complex which in turn facilitate EGFP transcription, pET21b-LasR-pUC57-pLasRL-EGFP was chosen to test the effect of transformation of different ratio of pET21b-LasR plasmid and pUC57-LasRL-EGFP plasmid into BL21 strain. The tested ratios were 1:1, 2:1 and 3:1. We found that 3:1 ratio of pET21b-LasR to pUC57-LasRL-EGFP gave the highest fluorescence signals. In the end, all four biosensors showed moderate green fluorescence signal after 15h as shown below.

**(A) OD600 and EGFP production of the three different ratios of the LasR sensing module and the EGFP reporting module in pET21b-LasR-pUC57-pLasRL-EGFP incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-6 over 3 hours**

**(i) 1:1 ratio**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-1.png)

**(ii) 2:1 ratio**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-2-new.png)

**(iii) 3:1 ratio**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-3-new.png)

The EGFP fluorescence intensity measured by the microplate reader was expressed in arbitrary unit as relative fluorescence unit (RFU). The PBS buffer was used to determine the background fluorescence intensity. The results showed that the EFGP production increase with time in all tested ratios of the LasR sensing module and the EGFP reporting module in pET21b-LasR-pUC57-pLasRL-EGFP. The fluorescence intensity of biosensors was ranged from 0.2138 RFU to 0.4387 RFU. Since the fluorescence signals were weak after 3h incubation with AHL cocktails, fluorescence images were taken after 15h incubation with AHL cocktails.

**(B) Fluorescence images of the three different ratios of the LasR sensing module and the EGFP reporting module in pET21b-LasR-pUC57-pLasRL-EGFP incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-6after 15 hours**

Incubation with distilled water was used as the negative control. The bright-field images showed the cell morphology of the biosensor cells and the fluorescence images showed green fluorescence signals emitted from the cells.

**(i) 1:1 ratio**

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0001.jpg)

**(ii) 2:1 ratio**

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0002.jpg)

**(iii) 3:1 ratio**

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0003.jpg)

The results showed that moderate green fluorescence signals were emitted from the cells in all tested ratios of the LasR sensing module and the EGFP reporting module in pET21b-LasR-pUC57-pLasRL-EGFP.

**(C) OD600 and EGFP production of the four biosensors incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-6M over 1.5 hours**

**(i) pET21b-LasR-pUC57-pLasRL-EGFP**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-4.png)

**(ii) pET21b-LasR-pUC57-pLasR3-EGFP**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-5.png)

**(iii) pET23b-LasR-pUC57-pLasRL-EGFP**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-6.png)

**(iv) pET23b-LasR-pUC57-pLasR3-EGFP**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-7.png)

The results showed that the EFGP production increase with time in all four biosensors. The fluorescence intensity of biosensors was ranged from 0.1344 RFU to 0.1780 RFU. Since the fluorescence signals were weak after 3h incubation with AHL cocktails, fluorescence images were taken after 15h incubation with AHL cocktails.

**(D) Fluorescence images of the biosensors incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-6M after 15 hours**

Incubation with distilled water was used as the negative control. The bright-field images showed the cell morphology of the biosensor cells and the fluorescence images showed green fluorescence signals emitted from the cells.

**(i) pET21b-LasR-pUC57-pLasRL-EGFP**

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0001.jpg)

**(ii) pET21b-LasR-pUC57-pLasR3-EGFP**

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0004.jpg)

**(iii) pET23b-LasR-pUC57-pLasRL-EGFP**

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0005.jpg)

**(iv) pET23b-LasR-pUC57-pLasR3-EGFP**

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0006-2.jpg)

The results showed that moderate green fluorescence signals were emitted from the cells in all four biosensors afte 15h incubation with AHL cocktails.

A sensitive biosensor should allow a rapid detection and monitoring of a relevant range of AHLs within samples. Our results indicated that the developed biosensors showed obvious fluorescence signals only after 15h and they were not sensitive enough to detect the presence of AHL molecules. Therefore, we adopted another approach to construct a new biosensor by integrate LasR and pLasRL-EGFP in a single plasmid (pSB1C3) instead of two separate plasmids. The resulting biosensor, named pSB1C3-LasR-pLasRL-EGFP showed high EGFP production rates in the presence of AHL cocktails within 3 hours which demonstrate the successful engineering of the biosensor.

**(E) OD600 and EGFP production of pSB1C3-LasR-pLasRL-EGFP incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-5M and 1x10-11M after 16 hours**

**(i) 1x10-5M**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-8.png)

**(ii) 1x10-11M**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-9.png)

The results showed that the EFGP production increase significantly with time. The fluorescence intensity of biosensors was ranged from 4.743 RFU to 48.74 RFU when incubated with 1x10-5M AHL cocktails and from 3.346 RFU to 16.16 RFU when incubated with 1x10-11M AHL cocktails after 16h. The highest fluorescence intensity of biosensors in the previous study was ranged from 0.2138 RFU to 0.4387 RFU when using different tested ratio of the LasR sensing module and the EGFP reporting module incubated with 1x10-6M AHL cocktail after 3h. In this study, the fluorescence intensity of biosensors was 5.959 RFU when incubated with 1x10-5M AHL cocktail after 2.5h. Thus, the new biosensors exhibited at least 10-fold (13.5-fold) higher fluorescence intensity compared with biosensors made in the previous study.

**(F) Fluorescence images of pSB1C3-LasR-pLasRL-EGFP incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-11M after 3 hours**

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0011-1.jpg)

These results demonstrated that a biosensor for the detection of a *P. aeruginosa*-specific quorum sensing molecules was successfully engineered and a strong GFP expression was developed within 3 hours after incubation with AHL molecules as low as 1x10-11M.

Future prospect

This engineering cycle has led us to further characterize the specificity of the engineered biosensor towards the three AHL molecules in the cocktail. Besides, we will study the ability of the biosensor to detect native AHL molecules produced by *P. aeruginosa*. Ultimately, we will replace the EGFP expression system in the reporting module with a reporter system based on pigment production such as red pigment lycopene as a visible readout for the development of paper-based device for microplastic detection.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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Content

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[* Transformation](#T)[* Bacterial DNA extraction(mini-prep)](#BE)[* Restriction enzyme digestion](#RED)[* PCR](#PCR)[* Gel electrophoresis](#GE)[* Preparation of N-acylhomoserine lactone (AHL) stock solutions](#PRE-N)[* AHL induction](#AHLI)[* Modifications of protocol](#MP)[* pSB1C3-LasR-LasRL-EGFP](#PSB)

Transformation

After receiving the gene synthesized by Synbio Technologies, 2μg of the powder in the vial was
dissolved with 20μL of MilliQ water, forming a solution with a concentration of 100μg/μL.
It is kept at -20°C. Bacterial tranformation was performed in order to produce multiple copies of
DNA for experiments.

* 1. Mix 1μL of the plasmid DNA with 200μL of *E. coli* DH5α cells.
* 2. Put the mixture in ice for 30 seconds.
* 3. Incubate at 42°C for 90 seconds.
* 4. Put in the ice again for 1-2 minutes.
* 5. Incubate at 37°C at 200rpm for 1 hour.
* 6. Discharge until 200μl of liquid is left, and pipette up and down
* 7. Spread plate

pET plasmid expressing LasR sensing module and pUC57 plasmid expressing pLasRL or pLasR3
were co-transformed into BL21C41(DE3)pLysS. In the transformation, 50 ng of each plasmid DN
A was used in 1:1 modules ratio. In 2:1 and 3:1 modules ratio, 100 ng and 150 ng of plasmid expr
essing sensing module were co-transformed with 50 ng of plasmid expressing reporting module res
pectively. The resulting engineered biosensors were named as pET21b-LasR-pUC57-pLasRL-EGFP, pET21b-LasR-pUC57-pLasR3-EGFP, pET23b-LasR-pUC57-pLasRL-EGFP and pET23b-LasR-pUC57-pLasR3-EGFP.

Each plasmid contains an antibiotic-resistant gene on it, whereas the antibiotic resistant genes of the 2 modules are different. The 2 antibiotics of the corresponding antibiotic-resistant gene were applied, indicating that only bacteria with both plasmids present can survive. The antibiotics we used were carbenicillin in pET plasmids and kanamycin in pUC57 plasmids. The resulting biosensor cells were selected in LB medium with both 100mg/ml carbenicillin (C) and 50mg/ml kanamycin (K).

Bacterial DNA extraction (mini-prep)

* 1. Centrifuge 1.5 mL of bacterial culture to form a pallet.
* 2. Discard supernatants.
* 3. Add 250 μL of the Resuspension Solution to resuspend the pellet. Vortex to ensure complete resuspension.
* 4. Add 250 μL of Lysis Buffer to lyse cells. Invert the tube 4-6 times. Incubate for 2 mins (incubation time should not exceed 5mins to avoid denaturation of supercoiled plasmid DNA).
* 5. Add 350 μL of Neutralization Buffer to neutralize the lysate. Invert the tube 4-6 times.
* 6. Centrifuged for 5 mins to pellet cell debris and chromosomal DNA.
* 7. Transfer supernatant to the GeneJET spin column by decanting or pipetting.
* 8. Centrifuge for 1 minute. Discard the flow-through and place the column back into the same collection tube.
* 9. Add 500 μL of Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Re-insert the column into the same collection tube.
* 10.Repeat step 9
* 11.Discard the flowthrough and centrifuge for an additional 1 min to remove the residual Wash Solution.
* 12.Transfer the GeneJET spin column into a fresh 1.5mL microcentrifuge tube. Add 30 μL of Milli-Q water to the center of the GeneJET spin column membrane to elute the plasmid DNA. Incubate for 2 minutes at room temperature and centrifuge for 2 min.
* 13.Discard the column and store the purified plasmid DNA at -20°C.

Restriction enzyme digestion:

![](https://static.igem.wiki/teams/4946/wiki/experiment-tables/re-digestion.png)

If there is only 1 enzyme, replace enzyme 2 with H2O
Incubate mix at 37°C for 30 min

PCR

![](https://static.igem.wiki/teams/4946/wiki/experiment-tables/master-mix-component.png)

* 1. Before the replication cycle had begun, the DNA samples were heated at 95°C for 5 minutes, and then the cycle began.
* 2. Reaction mixture was exposed to a high temperature of 95°C for 30 seconds to denature.
* 3. The samples were annealed at a lower temperature of 55°C for 30 seconds.
* 4. The temperature was then raised back up slightly to 72°C for 1 minute, completing one cycle of DNA duplication.
* 5. The cycle was conducted 30 times.
* 6. After the cycle, the DNA were incubated at 72°C for 5 minutes.
* 7. The DNA are stored at 4°C.

Gel electrophoresis

1% Gel

* 1. For each 100mL of gel, 1g of agarose powder is added to 100mL of 1XTBE buffer.
* 2. The mixture is then microwaved until the powder dissolved completely.
* 3. The mixture is then cooled down to room temperature under running water.
* 4. 1µL of midori green dye is added to the mixture.
* 5. The mixture is poured into the gel tank and a comb is inserted.
* 6. After the gel solidifies, it is put into the gel tank which is filled with 1X TBE buffer.
* 7. The comb is removed carefully to prevent the formation of air bubbles in the wells.
* 8. DNA ladder and samples are loaded into the wells.
* 9. A electric field is applied with anode at the side that the DNA samples run to.

Loading buffer is added to the DNA samples before loading.
DNA samples should be put in icebath to prevent degradation of samples.

Preparation of N-acylhomoserine lactone (AHL) stock solutions

We have ordered 3 synthetic AHL molecules from commercial companies, named N-butyryl L-homoserine lactone (C4HSL), N-3-oxo-decanoyl L-homoserine lactone (C10HSL) and N-3-oxo-dodecanoyl L-homoserine lactone (3OC12-HSL). They were first dissolved to produce a stock solution of 1x10-2M, by using the following equation:

![](https://static.igem.wiki/teams/4946/wiki/2023-10-09-163453.png)

![](https://static.igem.wiki/teams/4946/wiki/2023-10-09-163626.png)

1x10-2M is the stock solution for AHL. We carry out serial dilution and make AHL concentrations of 1x10-3M, until 1x10-11M.

When incubate AHL in bacterial cultures, we add AHL to bacterial culture in the ratio of 1:99 AHL to bacteria. The final concentration will be 100 times lower than the concentration of the AHL solution added.

AHL induction

Starter culture

1. For starter culture, a bacterial colony is picked from the bacterial plate and transferred into 1mL LKC medium.
2. Shake at 37°C at 230 rpm overnight.

Bacterial subculture

1. Carry out subculture by adding starter culture to LKC in the ratio of 1:999 starter culture to LKC in a 50 ml falcons. The final volume of the subculture is determined by the amount of culture we needed for the AHL induction experiment.
2. The falcon is put to shake at 33°C at 210 rpm.
3. It is allowed to grow for around 3 hours until the bacterial concentration reaches 0.3 OD600.
4. The culture in the falcon is aliquoted to different 15ml culture tubes, with AHL induced and no AHL added as a negative control. This is when 0h starts.
5. After collecting 300μL sample, the bacteria were put back into the shaker at 210 rpm.
6. Collection of samples takes place every half an hour until 3 hours.
7. At each time point, 1mL of culture is transferred to a cuvette for each culture tube, and bacterial concentration OD600 is measured using a NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer. 50μL of the sample is collected to be observed under a fluorescence microscope.

Washing cells with PBS (phosphate buffer saline)

PBS is an isotonic solution often used to wash cells to remove impurities.

1. Centrifuge at 14,000 rpm for 1 minute to form a pellet.
2. Discard the supernatant and add 500µL of 1x PBS to each Eppendorf
3. Vortex and centrifuge for 1 minute.
4. Repeat steps 2 and 3.
5. Add 200µL of 1x PBS to resuspend the cells, then pipette up and down to mix.

200uL of samples are added into a well on a 96 well plate, where the relative fluorescence unit (RFU) is measured by the microplate reader.

Modifications of protocol

**Construct**

We used different constructs to compare the use of different pET vectors 21b and 23b or the effect of different inducible promoters in pUC57-EGFP in the EGFP production of biosensors.

From interviews with professors, we acknowledged that in a situation where there is more AHL than LasR protein, the LasR protein formed may not be sufficient to form LasR-AHL complexes with AHL. LasR becomes a limiting factor. Thus, we tried to modify the constructs by using ratios of 2:1 and 3:1 (sensing module:reporting module) for constructs to test the effect of different ratios on the fluorescence signal exhibited by the constructs.

**AHL concentration**

We used various concentrations of AHL to investigate whether different AHL concentrations would lead to any correlation between fluorescence data. AHL concentrations we used ranged from 1x10-3M to 1x10-11M in cocktail.

**Autofluorescence (primary fluorescence)**

It is the fluorescence of naturally occurring substances. They can be detected under a fluorescence microscope and thus may affect our results.

We have changed dissolved AHL solutions with DMSO, originally carrying out serial dilution with DMSO to using MilliQ water. PBS is diluted without mixing precipitation calcium chloride when diluting. Both changes can reduce autofluorescence from other substances.

**Shaking speed**

We changed bacterial shaking speed from 210 rpm to 250 rpm to increase the rate of bacterial growth.

**Addition of IPTG**

We discovered that IPTG can activate pET vectors and promote translation. We added 7 μL IPTG per 3mL culture which results in concentration of 0.5 mM. Accordingly, we changed starting OD600 to 0.6 OD600 as it is the optimum value to best utilize IPTG.

**OD value**

We started our experiments at 0.3 OD600. We later discovered that OD 0.3 was not optimal for AHL induction as the rate of cell growth was too slow. OD 0.5 is farther along the exponential phase of growth. The bacterial exponential growth phase is towards 1 OD600 and thus 0.5 OD600 is more optimal than OD 0.3.
We later introduced the addition of IPTG, thus changing starting OD600 from 0.5 to 0.6 which is the optimum OD600 for IPTG to work most efficiently.

**Negative experiments**

We designed four negative experiments to investigate the autofluorescence of substances throughout the experiment.

We measured the RFU of PBS, milliQ and DMSO of different concentrations, LKC medium and LCM medium. Such negative experiments show the fluctuations of autofluorescence in these substances so as to compare with fluorescence levels the construct produces. It shows the effect of DMSO in increasing autofluorescence and that it will affect fluorescence value measured in the construct . Results also show that LKC has high RFU and may affect the result during quantifying EGFP from the construct.

**Fluorescein**

We also used a fluorescence chemical named fluorescein. We obtained the optimal concentration by serial dilution and then measured its RFU under the microplate reader in order to calibrate it. Fluorescein can act as a positive control as it shows the most optimal situation where constant fluorescence is emitted.

1. Get the stock reagent tube with Fluorescein calibrant from the Measurement Kit. It is in powder form. Spin down to make sure the pellet is at the bottom.
2. Transfer 1.0 mL of Phosphate Buffered Saline (PBS) to stock reagent tube Fluorescein calibrant. Resuspend pipetting up and down a few times, and vortex for 30 seconds. This is now the reconstituted Stock Fluorescein Solution 10X with a concentration of 100 μM in PBS.
3. Obtain a tube to make the working concentration solution 1X Fluorescein.
4. Dilute the Stock Fluorescein Solution 10X with 1X PBS to make a 1X reference working solution with a concentration of 10 uM. E.g. dilute 100 μL of 10X fluorescein stock into 900 μL 1X PBS.

Serial Dilution5. Prepare a 96 well microplate.
6. Transfer 100.0 μL of PBS to wells A2:B12 of calibration plate. Column 12 has the blanks.
7. Transfer 200.0 μL of Fluorescein 1X solution to wells A1 and B1 of calibration plate.
8. Perform a series of 10 2-fold dilutions from the first column of the calibration plate using PBS or Water (already prefilled) as diluent to a final volume of 200.0uL in wells A1:B11 of the calibration plate. For each of the ten steps transfer 100uL from the initial column into the next one and pipette up and down 3X to ensure the dilution is mixed homogeneously before the next transfer.
9. Discard 100.0μL from wells A11:B11 of calibration plate. This step ensures that all wells contain the same volume (100μL).
10. Transfer 100.0μL of PBS to wells A1:B12 of calibration plate. This will bring all wells to volume 200μL. Pipette up and down 3X to ensure the dilution is mixed homogeneously.

**J364000**

We measured fluorescence of Part: J364000 which is a GFP expressing constitutive device for the 2017 iGEM InterLab study. This construct can constantly produce the GFP protein. We can quantify the fluorescence of this gene using the microplate reader.
It can act as a positive control as it shows the most optimal situation where constant fluorescence is emitted.
We measured the RFU value of J364000 under a microplate. We added 200uL of control LCM into wells A1-C1. We added 200uL culture of colony 1 of J364000 and colony 2 into wells A2-C2 and A3-C3 respectively.

**Microplate**

Experiments show that the fluorescence emitted from the EGFP protein produced after washing cells was not up to standard; and that LKC used in washing cells with 1x PBS had higher autofluorescence. We then modified the experiment protocol where we directly measure RFU and OD value of constructs in the microplate reader.
Subculture protocol is the same as previously mentioned.
After subculture, add 2μL subculture and 2ml LKC/LCM.Then, add 20μL AHL + 5μL IPTG into the culture.
Then, extract 200 μL from the culture and add into microplate wells. Add positive control into wells A-C:9-12. Add negative control into wells A-C:1-4.
Reserve one row for control which consists of control LKC in wells D1-8 and LCm in wells 9-12.
The rfu data obtained will include autofluorescence from impurities or fluorescence from other solutions used during experiment. We can obtain the rfu produced by the construct by minusing the rfu from the construct to the control.

pSB1C3-LasR-LasRL-EGFP

pSB1C3-LasR-LasRL-EGFP is a recombinant plasmid that contains both our sensing and reporting module. It consists of a promoter followed by RBS and LasR gene and a terminator to produce LasR protein. This is followed by the pLasRL inducible promoter with an RBS, the EGFP gene and a terminator. LasR protein binds with AHL molecules to form a LasR-AHL complex. Then, the complex binds with a pLasRL promoter with RBS (Part: B0034) to activate the EGFP gene to express EGFP.
Transformation, Bacterial DNA extraction, restriction enzyme digestion, PCR, gel electrophoresis, was carried out in the same way as the former constructs.
Transformation
1μL of the pSB1C3-LasR-pLasRL-EGFP was transformed into the host cell E. Coli strain DH5-alpha, C41(DE3) by heat shock method. The resulting biosensor cells were selected in LB medium containing 34mg/ml chloramphenicol (CM).

**Starter culture**

1. For starter culture, a bacterial colony is picked out from the bacterial plate and transferred into 1mL LCM.
2. Shake at 37°C at 230 rpm overnight.

**Subculture:**

1. Carry out subculture by adding starter culture to LCM in the ratio of 1:499 starter culture to LKC in a falcon. The final volume of the subculture is determined by the amount of culture we needed for the AHL induction experiment.
2. The falcon is put to shake at 37°C at 230 rpm
3. It is allowed to grow for 3 hours until the bacterial concentration reaches 0.6 OD600.
4. The culture in the falcon is aliquoted to different culture tubes. Different concentrations of AHL are added. This is when 0h starts.
5. After collecting the sample, the bacteria were put back into the shaker at 250 rpm
6. Collection of samples takes place every hour until 3 hours or assigned time points.
7. At each time point, 1mL of culture is transferred to a cuvette for each culture tube, and bacterial concentration OD600 is measured using a NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer.

**AHL induction**

Collect 300 uL of solution from culture tube to undergo cell washing protocol.

1. Centrifuge at 14,000rpm for 1 minute to form a pellet.
2. Discard the supernatant and add 500µL of 1x PBS to each Eppendorf
3. vortex and centrifuge for 1 minute.
4. Repeat steps 2 and 3.
5. Discard supernatants. Add 200µL of 1x PBS and pipette up and down to resuspend the cell

Samples are added into a well on the microplate, where the relative fluorescence unit (RFU) is measured by the microplate reader.
Blank: 1x PBS

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

Microplastics has been identified as hubs for microbial pathogens and their antimicrobial resistance (AMR) due to hydrophobicity, greater surface area and pit forming nature of microplastics. These pathogenic bacteria form biofilms on the microplastics. Biofilm formation is governed by the quorum sensing (QS) genes expressed as a result of the accumulation of a critical cell density. *P. aeruginosa* is a common *Pseudomonas* sps attach to the microplastics and contribute to microplastic-associated AMR. In our project, we make use of the complete quorum sensing regulatory circuit LasI-LasR in *P. aeruginosa* to engineer a whole cell biosensor based on the LasI-LasR system to detect and quantitatively measure the presence of quorum sensing molecules secreted from *P. aeruginosa* for detection of microplastic pollution levels in water samples. Below are the important findings of our projects.

1. Confirmation of the presence of DNA inserts in plasmid constructs

We integrate the LasR sensing module and the reporting module that expressed EGFP to demonstrate the sensitivity of the biosensor in response to the quorum-sensing molecules of the *P. aeruginosa*. The transcription factor LasR was expressed under the control of the constitutive T7 promoter in the plasmid pET 21b and pET 23b. Two pET plasmids were used in order to compare if the presence of lac operator in pET 21a can prevent leaky expression of the LasR and thus give less background fluorescence signals in the uninduced state. Besides, we used two LasR inducible promoters, pLasRL and pLasR3 in the reporting module in order to compare the two promoter strengths. The two modules were transformed into DH5α individually. The extracted DNA of the two modules were then co-transformed into the BL21 C41(DE3)pLysS. The resulting engineered biosensors were named as pET21b-LasR-pUC57-pLasRL-EGFP, pET21b-LasR-pUC57-pLasR3-EGFP, pET23b-LasR-pUC57-pLasRL-EGFP and pET23b-LasR-pUC57-pLasR3-EGFP. PCR was performed to confirm the presence of LasR, pLasRL and pLasR3 in the transformed colonies.

![](https://static.igem.wiki/teams/4946/wiki/gel-image/1.jpg)

###### Figure 1. Colony PCR screening of recombinant pET-LasR plasmids transformed in DH5α competent cells.

![](https://static.igem.wiki/teams/4946/wiki/gel-image/2.jpg)

###### Figure 2. Colony PCR screening of pUC57-pLasR plasmids transformed in DH5α competent cells.

Figure 1 and Figure 2 showed that PCR products with the correct size of 566 bp in LasR,
274 bp in pLasRL and 307 bp in pLasR3 were amplified in five colonies of all constructs confirming the presence of LasR, pLasRL and pLasR3 in the expression vector pET 21b,
pET 23b and pUC57.

![](https://static.igem.wiki/teams/4946/wiki/gel-image/3-2.jpg)

###### Figure 3. Colony PCR screening of pET23b-LasR-pUC57-pLasR3-EGFP plasmid transformed in BL21 C41(DE3) pLysS competent cells.

Figure 3 showed that PCR products with the correct size of 566 bp in LasR and 307 bp in pLasR3 were amplified in five colonies of the construct confirming the presence of LasR and pLasR3 in the pET23b-LasR-pUC57-pLasR3-EGFP plasmid.

![](https://static.igem.wiki/teams/4946/wiki/gel-image/4.jpg)

###### Figure 4. Colony PCR screening of pET21b-LasR-pUC57-pLasRL-EGFP and pET23b-LasR-pUC57-pLasRL-EGFP plasmids transformed in BL21 C41(DE3) pLysS competent cells.

Figure 4 showed that PCR products with the correct size of 566 bp in LasR were amplified in four and eight colonies of the constructs confirming the presence of LasR in the
pET21b-LasR-pUC57-pLasRL-EGFP and pET23b-LasR-pUC57-pLasRL-EGFP plasmids.

![](https://static.igem.wiki/teams/4946/wiki/gel-image/5.jpg)

###### Figure 5. Colony PCR screening of pET21b-LasR-pUC57-pLasRL-EGFP and pET23b-LasR-pUC57-pLasRL-EGFP plasmids transformed in BL21 C41(DE3) pLysS competent cells.

![](https://static.igem.wiki/teams/4946/wiki/gel-image/6.jpg)

###### Figure 6. Colony PCR screening of pET21b-LasR-pUC57-pLasR3-EGFP plasmid transformed in BL21 C41(DE3) pLysS competent cells.

Figure 6 showed that PCR products with the correct size of 566 bp in LasR and 307 bp in pLasR3 were amplified in seven colonies of the construct confirming the presence of LasR and pLasR3 in the pET21b-LasR-pUC57-pLasR3-EGFP plasmid.

2. EGFP production of the three different ratios of pET21b-LasR-pUC57-pLasRL-EGFP

The engineered biosensor is composed of two modules: the sensing module and the reporting module. In the LasR quorum-sensing system, the sensing module expresses the transcription factor, LasR which can bind to the AHL moleucles. The formation of LasR-AHL complex can then bind to an inducible promoter, pLasRL or pLasR3 which lead to the activation of the reporting module to produce green fluorescence readout. Since the green fluorescence signals emitted by the biosensor cells were weak (data not shown), we tried different ratio of LasR sensing module to pLasRL-EGFP reporting module as one of the modifications in the protocol. We hypothesized that the sensing module in the expression plasmids need to be in excess so that more AHL molecules can bind and drive the expression of reporting module. We transformed three different ratios of the LasR sensing module and the EGFP reporting module in pET21b-LasR-pUC57-pLasRL-EGFP construct including 1:1, 2:1 and 3:1. The biosensor cells were incubated with a cocktail of synthetic AHL molecules, N-butyryl L-homoserine lactone (C4HSL), N-3-oxo-decanoyl L-homoserine lactone (C10HSL) and N-3-oxo-dodecanoyl L-homoserine lactone (3OC12-HSL) at a concentration of 1x10-6M over 3 hours. 1x10-6M AHL falls within the relevant concentrations of AHL concentrations (1x10-8 to 5x10-6 for 3OC12-HSL) in water environments as reported in the literature[1].

**(i) 1:1 ratio**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-1.png)

**(ii) 2:1 ratio**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-2-new.png)

**(iii) 3:1 ratio**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-3-new.png)

###### Figure 7. OD600 and EGFP production of pET21b-LasR-pUC57-pLasRL-EGFP containing the LasR sensing module and pLasRL-EGFP reporting module in a (i) 1:1; (ii) 2:1; (iii) 3:1 ratio.

Readings of OD600 were shown to indicate the normal cell growth over 3h of AHL cocktails incubation. The EGFP fluorescence intensity measured by the microplate reader was expressed in arbitrary unit as relative fluorescence unit (RFU). The PBS buffer was used to determine the background fluorescence intensity. The results showed that the EFGP production increase with time in all tested ratios of the LasR sensing module and the EGFP reporting module in pET21b-LasR-pUC57-pLasRL-EGFP. The fluorescence intensity of biosensors was similar and it ranged from 0.2138 RFU to 0.4387 RFU.

3. Fluorescence images of the three different ratios of pET21b-LasR-pUC57-pLasRL-EGFP

Since the fluorescence signals were weak after 3h incubation with AHL cocktails, fluorescence images were taken after 15h incubation with AHL cocktails.

**(i) 1:1 ratio**

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0001.jpg)

**(ii) 2:1 ratio**

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0002.jpg)

**(iii) 3:1 ratio**

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0003.jpg)

###### Figure 8. Fluorescence microscope images of pET21b-LasR-pUC57-pLasRL-EGFP containing the LasR sensing module and pLasRL-EGFP reporting module in a (i) 1:1; (ii) 2:1; (iii) 3:1 ratio. Incubation with distilled water was used as the negative control. The bright-field images showed the cell morphology of the biosensor cells and the fluorescence images showed green fluorescence signals emitted from the cells.

Figure 8 showed that moderate green fluorescence signals were emitted from the cells in all tested ratios of the LasR sensing module and the EGFP reporting module in pET21b-LasR-pUC57-pLasRL-EGFP. Also, there was no significant difference between the different ratios of the sensing module and the reporting module and EGFP production.

4. EGFP production of the four engineered biosensors

The engineered biosensors were incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-6M for 1.5 hours.
Readings of OD600 were shown to indicate the normal cell growth over 1.5h of AHL cocktails incubation. The EGFP fluorescence intensity measured by the microplate reader was expressed in arbitrary unit as relative fluorescence unit (RFU). The PBS buffer was used to determine the background fluorescence intensity.

**(i) pET21b-LasR-pUC57-pLasRL-EGFP**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-4.png)

**(ii) pET21b-LasR-pUC57-pLasR3-EGFP**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-5.png)

**(iii) pET23b-LasR-pUC57-pLasRL-EGFP**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-6.png)

**(iv) pET23b-LasR-pUC57-pLasR3-EGFP**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-7.png)

###### Figure 9. OD600 and EGFP production of the four engineered biosensors (i) pET21b-LasR-pUC57-pLasRL-EGFP; (ii) pET21b-LasR-pUC57-pLasR3-EGFP; (iii) pET23b-LasR-pUC57-pLasRL-EGFP and (iv) pET23b-LasR-pUC57-pLasRL-EGFP

The results showed that the EFGP production increase with time in all four biosensors. The fluorescence intensity of biosensors was similar and it ranged from 0.1344 RFU to 0.1780 RFU.

5. Fluorescence microscope images of the four engineered biosensors

Since the fluorescence signals were weak after 1.5h incubation with AHL cocktails, fluorescence images were taken after 15h incubation with 1x10-6M AHL cocktails.

**(i) pET21b-LasR-pUC57-pLasRL-EGFP**

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0001.jpg)

**(ii) pET21b-LasR-pUC57-pLasR3-EGFP**

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0004.jpg)

**(iii) pET23b-LasR-pUC57-pLasRL-EGFP**

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0005.jpg)

**(iv) pET23b-LasR-pUC57-pLasR3-EGFP**

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0006-2.jpg)

###### Figure 10. Fluorescence microscope images of the four biosensors. (i) pET21b-LasR-pUC57-pLasRL-EGFP; (ii) pET21b-LasR-pUC57-pLasR3-EGFP; (iii) pET23b-LasR-pUC57-pLasRL-EGFP; (iv) pET23b-LasR-pUC57-pLasR3-EGFP. Incubation with distilled water was used as the negative control. The bright-field images showed the cell morphology of the biosensor cells and the fluorescence images showed green fluorescence signals emitted from the cells.

The results showed that moderate green fluorescence signals were emitted from the cells in all four biosensors after 15h incubation with 1x10-6M AHL cocktails. Among the four biosensors, pET21b-LasR-pUC57-pLasRL-EGFP showed more obvious green fluorescence signals with less autofluorescence as shown in Figure 9(i). However, our results showed that there was no significant difference in EGFP production among the four biosensors indicating that the uses of either pET 21b and pET 23b and either pLasRL and pLasR3 in the expression plasmid exhibit no differences in the functionality of engineered biosensors.

A sensitive biosensor should allow a rapid detection and monitoring of a relevant range of AHLs within samples. Our results indicated that the developed biosensors showed moderate fluorescence signals after 15h and they were not sensitive enough to detect the presence of AHL molecules. Therefore, we adopted another strategy to construct a new biosensor by integrate the LasR sensing module and the pLasRL-EGFP reporting module in a single plasmid (pSB1C3) and then transform into BL21 strain instead of transform two separate plasmids into a BL21 strain. After confirmation of the presence of insert DNA, the resulting biosensor, named pSB1C3-LasR-pLasRL-EGFP was tested for the specificity and sensitivity.

6. Confirmation of the presence of DNA inserts in pSB1C3-LasR-pLasRL-EGFP

![](https://static.igem.wiki/teams/4946/wiki/after-wiki-freeze/gel-psb1c.png)

###### Figure 11. Colony PCR screening of pSB1C3-LasR-pLasRL-EGFP plasmid transformed in BL21 competent cells.

Figure 11 showed that PCR products with the correct size of 566 bp in LasR and 274 bp in pLasRL were amplified in five colonies of the construct confirming the presence of LasR and pLasRL in the pSB1C3-LasR-pLasRL-EGFP plasmid.

7. Fluorescence microscope images of three colonies of pSB1C3-LasR-pLasRL-EGFP

Three colonies of the biosensor were incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-11M for 3 hours and the fluorescence microscope images were taken.

**(i) pSB1C3-LasR-pLasRL-EGFP - 1**

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0007.jpg)

**(ii) pSB1C3-LasR-pLasRL-EGFP - 3**

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0008.jpg)

**(iii) pSB1C3-LasR-pLasRL-EGFP - 4**

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0009.jpg)

###### Figure 12. Fluorescence microscope images of three colonies of pSB1C3-LasR-pLasRL-EGFP. (i) pSB1C3-LasR-pLasRL-EGFP-1; (ii) pSB1C3-LasR-pLasRL-EGFP-3; (iii) pSB1C3-LasR-pLasRL-EGFP-4.

These results demonstrated that a more sensitive biosensor for the detection of a *P. aeruginosa*-specific quorum sensing molecules was successfully engineered and a strong GFP expression was developed within 3 hours after incubation with AHL molecules as low as 1x10-11M. Among the three colonies, pSB1C3-LasR-pLasRL-EGFP-4 showed the highest fluorescence signals with the least background, thus it was used for subsequently studies.

8. EGFP production of pSB1C3-LasR-pLasRL-EGFP

A. EGFP production of pSB1C3-LasR-pLasRL-EGFP incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-5M and 1x10-11M in 16 hours

**(i) 1x10-5M**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-8.png)

**(ii) 1x10-11M**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-9.png)

###### Figure 13. EGFP production of pSB1C3-LasR-pLasRL-EGFP incubated with a cocktail of synthetic AHL molecules at a concentration of 1x10-5M and 1x10-11M in 16 hours

The results showed that the EFGP production increase significantly with time. The fluorescence intensity of biosensors was ranged from 4.743 RFU to 48.74 RFU when incubated with 1x10-5M AHL cocktails and from 3.346 RFU to 16.16 RFU when incubated with 1x10-11M AHL cocktails after 16h. The highest fluorescence intensity of biosensors in the previous study was ranged from 0.2138 RFU to 0.4387 RFU when using different tested ratio of the LasR sensing module and the EGFP reporting module incubated with 1x10-6M AHL cocktail after 3h. In this study, the fluorescence intensity of biosensors was 5.959 RFU when incubated with 1x10-5M AHL cocktail after 2.5h. Thus, the new biosensors exhibited at least 10-fold (13.5-fold) higher fluorescence intensity compared with biosensors made in the previous study.

B. EGFP production of pSB1C3-LasR-pLasRL-EGFP incubated with individual synthetic AHL molecules at a concentration of 1x10-7M in 4 hours

**(i) Distilled water (negative control)**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-10-new-new.png)

**(ii) N-butyryl L-homoserine lactone (C4HSL)**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-11-new.png)

**(iii) N-3-oxo-decanoyl L-homoserine lactone (C10HSL)**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-12-new.png)

**(iv) N-3-oxo-dodecanoyl L-homoserine lactone (3OC12-HSL)**

![](https://static.igem.wiki/teams/4946/wiki/data-chart/data-13-new.png)

###### Figure 14. EGFP production of pSB1C3-LasR-pLasRL-EGFP incubated with individual synthetic AHL molecules at a concentration of 1x10-7M in 4 hours. (i) Distilled water (negative control); (ii) N-butyryl L-homoserine lactone (C4HSL); (iii) N-3-oxo-decanoyl L-homoserine lactone (C10HSL); (iv) N-3-oxo-dodecanoyl L-homoserine lactone (3OC12-HSL)

EGFP production of biosensor cells were incubated with 1x10-7M N-butyryl L-homoserine lactone (C4HSL) was similar to that of negative control suggesting that the biosensor was unable to detect C4HSL. However, when the biosensor cells were incubated with 1x10-7M N-3-oxo-decanoyl L-homoserine lactone (C10HSL) and N-3-oxo-dodecanoyl L-homoserine lactone (3OC12-HSL), EGFP production increased up to 14.86 RFU. The results suggested that the biosensor was able to detect C10HSL and 3OC12-HSL.

9. Fluorescence microscope images of pSB1C3-LasR-pLasRL-EGFP after incubation with individual AHLs

![](https://static.igem.wiki/teams/4946/wiki/f-mic-photos/f-mic-photos/0011-1.jpg)

###### Figure 15. Fluorescence microscope images of pSB1C3-LasR-pLasRL-EGFP incubated with individual synthetic AHL molecules (C4HSL, C10HSL and 3OC12-HSL) at a concentration of 1x10-7M in 3 hours.

In the presence of the 1x10-7 C10HSL and 3OC12-HSL, the engineered biosensor cells produced strong green fluorescence signals while in the presence of C4HSL and in the control set-up, there was no green fluorescence signals observed. With strong GFP fluorescence signals produced by biosensor cells in the presence of C10HSL and 3OC12-HSL, the results suggested that the biosensor was able to detect C10HSL and 3OC12-HSL.

10. Response of pSB1C3-LasR-pLasRL-EGFP toward three synthetic AHLs

![](https://static.igem.wiki/teams/4946/wiki/charts/chart-1.png)

###### Figure 16. Response of pSB1C3-LasR-pLasRL-EGFP toward three individual synthetic AHL molecules at 1x10-6M and 1x10-7M C4HSL, C10HSL and 3OC12-HSL for 3h.

The biosensor was incubated in the presence of each AHL at 1x10-6M and 1x10-7M for 3h. The results are the means of triplicate experiments. The biosensor without the addition of AHL molecules was used as the negative control. The results demonstrated that the EGFP production rate was significantly higher in 1x10-7M C10HSL than that induced by the concentration of 1x10-6M C10HSL and induced by the addition of 3OC12-HSL. There was no EGFP production when the biosensor was induced by the addition of C4HSL.

11. EGFP production of pSB1C3-LasR-pLasRL-EGFP

![](https://static.igem.wiki/teams/4946/wiki/charts/chart-2.png)

###### Figure 17. EGFP production of pSB1C3-LasR-pLasRL-EGFP toward three individual synthetic AHL molecules at 1x10-7M C4HSL, C10HSL and 3OC12-HSL for 4h. Distilled water was used as a negative control.

The biosensor cells were incubated with 1x10-7M individual AHLs for 4h. The results are the means of triplicate experiments. It demonstrated that EGFP production rate of the biosensor was the highest when it was incubated with 3OC12-HSL followed by C10HSL. The biosensor did not show response when it was incubated with C4HSL.

12. EGFP production rates of pSB1C3-LasR-pLasRL-EGFP at different concentrations of synthetic AHL molecules 3OC12-HSL

We then characterized the sensitivity of the biosensor by quantifying the EGFP production rate in response to a range of AHL molecules, 3OC12-HSL from 1x10-7 to 1x10-14M for 19h. The results are the means of triplicate experiments.

![](https://static.igem.wiki/teams/4946/wiki/charts/chart-3.png)

###### Figure 18. EGFP production of pSB1C3-LasR-pLasRL-EGFP at 1x10-7M 3OC12-HSL for 5h.

The result demonstrated that the EGFP production rates of the biosensor started to increase when the concentration of 3OC12-HSL increased from 1x10-12M and peaked at a steady rate when the concentration of 3OC12-HSL reached 1x10-7M.

13. Quantitative relationship between the EGFP production rate and
3OC12-HSL concentration

![](https://static.igem.wiki/teams/4946/wiki/charts/chart-4.png)

###### Figure 19. The relationship between the EGFP production rate and different concentration of 3OC12-HSL.

An empirical mathematical model, the Hill equation was used to model EGFP production rate (y) as a function of the initial concentration of the synthetic AHL molecule, 3OC12-HSL. In the Hill equation[2]:

![](https://static.igem.wiki/teams/4946/wiki/charts/hill-equation.png)

Four parameters (A, B, C and n) were used to make the non-linear best fit curve using the experimental results. The parameter A is the baseline of EGFP production rate when AHL initial concentration is 0 M. The parameter B is the maximum EGFP production rate. The parameter C is called EC50 which represents the AHL concentration that can induce a half-maximal EGFP production rate.

14. Quantitative relationship between the EGFP production rate and
C10HSL concentration

![](https://static.igem.wiki/teams/4946/wiki/after-wiki-freeze/c10-chart-new-copy.png)

###### Figure 20. The relationship between the EGFP production rate and different concentration of C10-HSL.

The Hill equation showed that the biosensor was highly sensitive to the 3OC12-HSL with EC50 values as low as 2.823 x 10-11M 3OC12-HSL. The biosensor also sensitive to the C10HSL with EC50 values as low as 6.841 x 10-9M. It was shown that the concentration of
3OC12-HSL detected in the effluent from *P. aeruginosa* biofilm was 1.4 x 10-8M and its concentration within the biofilm was over 600 μM[3]. Also the concentration of 3OC12-HSL detected in the supernatant of planktonic cultures are estimated to be between 1 x 10-8 and 5 x 10-6M[4]. Our engineered biosensor with an EC50 value of 2.823 x 10-11M indicate the feasibility of the biosensor in detecting the 3OC12-HSL in both biofilm and water samples. Besides, the engineered biosensor had an EC50 value which was lower than the whole-cell biosensor that had an EC50 value of 5.91 x 10-9M for 3OC12-HSL used for detection of water contamination by *P. aeruginosa*[2]. This indicates the high sensitivity of our engineered biosensor in nanoscale in the detection of 3OC12-HSL.

Taken together, we first design and engineered four biosensors based on LasI-LasR quorum sensing regulatory circuit LasI-LasR in *P. aeruginosa*. The plasmids expressing the LasR sensing module and the pLasRL/pLasR3-EGFP reporting module were transformed into BL21 strain to yield the whole-cell biosensor with green fluorescence signal. With several modifications in the protocol, moderate green fluorescence signals were obtained in all four biosensors after 15h incubation with 1x10-6M AHL cocktails. No significant difference in the EGFP production in all biosensors. In an attempt to engineer a more sensitive biosensor with fast detection of AHL molecules in a sample, the strategy of integrating both the LasR sensing module and the pLasRL-EGFP in a single expression plasmid was used. The engineered biosensor with both modules integrated in a single expression plasmid was shown to induce a higher EGFP production rate at a lower AHL concentration and within a shorter time than the four biosensors with the two modules expressed in different plasmids. To conclude, a biosensor for the detection of a *P. aeruginosa*-specific quorum sensing molecules was successfully engineered and a strong GFP expression was developed within 3 hours in the presence of quorum sensing molecules of *P. aeruginosa*, 3OC12-HSL and C10HSL as low as 1x10-11M. In addition, the engineered biosensor had high sensitivity in response to the quorum sensing molecules, 3OC12-HSL with an EC50 value of 2.823 x 10-11M and C10HSL with EC50 values as low as 6.841 x 10-9M. The biosensor has potential applications to provide a rapid, sensitive and quantitative detection of the microplastic pollution levels in water samples.

Future perspectives

From literature reviews, we found that the engineering of whole-cell biosensors based on quorum sensing systems is mainly for biomedical application while the potential of biosensors for detection of microplastic level is very limited. For example, an *E. coli* biosensor which specifically detect *Vibrio cholerae* based on its quorum sensing molecule autoinducer-I (CAI-1) was developed to inhibit the growth of V. cholerae cells [5]. Besides, a whole-cell biosensor based on *P. aeruginosa* quorum sensing circuit was developed to detect short- and long-chain AHLs in sputum samples from cystic fibrosis lungs [6]. Recently, a whole-cell biosensor based on QscR quorum sensing signal system of *P. aeruginosa* was first developed to detect waterborne bacterial pathogens[2]. Thus, we aimed to apply *P. aeruginosa* LasI-LasR circuit to engineer biosensor for the detection of specific AHL molecules. The biosensor will ultimately use to quantitatively measure the presence of AHL molecules in water samples for monitoring microplastic pollution.

Our engineered biosensor had high sensitivity in response to the quorum sensing molecules, 3OC12-HSL, which is the cognate signal molecule of *P. aeruginosa*. To test the possibility of the use of the biosensor for detection of other AHL molecules, the specificity of the engineered biosensor toward N-butyryl L-homoserine lactone (C4HSL) and N-3-oxo-decanoyl L-homoserine lactone (C10HSL) were also be studied. The results demonstrated that the engineered biosensor had no response to C4HSL. However, the biosensor had a strong response to C10HSL as shown in Figure 16, the quantitative relationship between the EGFP production rate and C10HSL concentration showed EC50 values as low as 6.841 x 10-9M. C10HSL is one of the quorum sensing molecules secreted by key biofilm-forming bacteria on microplastics in marine environment [7]. The ability of the biosensor to respond to C10HSL is important for monitoring microplastics pollution as some biofilm-forming bacteria on microplastics produce C10HSL as the main quorum sensing signals. Also, we will further characterize the ability of the biosensor to detect native AHL molecules produced by *P. aeruginosa*.

For detection of environmental water samples, it is important to utilize a type of sensor and detection method which can be portable, that is to be used in the field site. Bacterial whole-cell biosensors have several advantages on the use because they are relatively easy and inexpensive to prepare and store, their ability to withstand a range of environmental conditions such as pH or temperature fluctuations and can be integrated into various platforms for rapid high-throughput or on-site detection. In this study, the highly efficient expression and easy detection of the green fluorescence signal was used to facilitate the characterization and optimization of the biosensor system. Ultimately, we will replace the EGFP expression system in the reporting module with a reporter system based on pigment production such as red pigment lycopene or colour production such as detection of blue β-galactosidase activity as a visible readout for the development of paper-based device for microplastic detection.

Whole-cell biosensors give promising results in the field with several advantages, but there are concern about the biosafety of using these systems in the field directly because this system is classified as genetically modified organisms. We have adopted the following strategies in project design to minimize the risks. First, we use plasmids as vectors to carry quorum sensing circuits rather than introducing the genome to inhibit the spread of recombinant DNA to environment and other organisms via Horizontal Gene Transfer (HGT). Besides, we add LasR regulatory elements, pLasRL and pLasR3 to the system to control the gene expression.

Engineered whole-cell biosensors with implemented quorum sensing circuit represents a good alternative for environmental monitoring of microplastics pollution. Also, the information of molecules to agonize and antagonize quorum-sensing systems provide further insights about bacterial communication.

Reference

1. Pearson, J.P., et al., A second N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa. Proceedings of the National Academy of Sciences, 1995. 92(5): p. 1490-1494.

2. Wu, Y., et al., A Whole-Cell Biosensor for Point-of-Care Detection of Waterborne Bacterial Pathogens. ACS Synthetic Biology, 2021. 10(2): p. 333-344.

3. Charlton, T.S., et al., A novel and sensitive method for the quantification of N-3-oxoacyl homoserine lactones using gas chromatography-mass spectrometry: application to a model bacterial biofilm. Environ Microbiol, 2000. 2(5): p. 530-41.

4. Hogan, D.A., A. Vik, and R. Kolter, A Pseudomonas aeruginosa quorum-sensing molecule influences Candida albicans morphology. Mol Microbiol, 2004. 54(5): p. 1212-23.

5. Jayaraman, P., et al., Repurposing a Two-Component System-Based Biosensor for the Killing of Vibrio cholerae. ACS Synth Biol, 2017. 6(7): p. 1403-1415.

6. Middleton, B., et al., Direct detection of N-acylhomoserine lactones in cystic fibrosis sputum. FEMS Microbiol Lett, 2002. 207(1): p. 1-7.

7. Xu, X., et al., Quorum sensing bacteria in microplastics epiphytic biofilms and their biological characteristics which potentially impact marine ecosystem. Ecotoxicology and environmental safety, 2023. 264: p. 115444.

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