Designed by: Fan Jiayu   Group: iGEM21_BS_United_China   (2021-10-03)

What it is? Why it is necessary?

Microorganisms often persist in their natural niches by attaching to surfaces and forming complex, sessile microbial communities known as biofilms. P. aeruginosa is one of the most common food pathogens which is able to form biofilms either independently by itself or jointly with other microorganisms. When the concentration of quorum sensing (QS) molecule, AHL, secreted by pathogens in the environment is above the threshold, P. aeruginosa will be coordinated by QS signal to construct the biofilms. AHL is the key signaling molecular and can initiate the bio-toxin production. Thus, we need a quick methods to detect the accumulation of AHL in contaminated food.

What it does?

Our E. bsuahlscout bio-brick contains 3 key elements including LuxR, QS promoter, and mCherry. LUXR is a protein coded by LuxR gene. The LuxR gene is regulated by T7 promoter, Lac operator and T7 terminator. The LUXR protein can combine the AHL to formate a LUXR-AHL complex which can bind to the QS promoter and recruit RNA polymerase to start the down-stream gene expression. QS promoter is a DNA element to react with LUXR-AHL complex. The mCherry gene is down-stream report gene of QS promoter and codes a red fluorescence protein.

How to use it?

E. bsuahlscout bio-brick can transform to any competent bacterial. We use E. Coli BL21 as a recipe and safe bacterial to do the transformation. We use the LB media to culture the E. bsuahlscout. When the OD value reach to 0.2-0.4, we add 200 uM IPTG into the LB media to activate the E. bsuahlscout bio-brick inside at 30 degree centigrade for 4 h. After that, we make the AHL detecting kit and 1 cube is from LB-agar plate which contains 500 uM IPTG. The sample LB media which contains AHL will culture the activated E. bsuahlscout at 30 about 1-2 hours until the red color show up.

Experiment results of Part BBa K3882000:E.bsuahlscout

1. Induce part1 by using the verdigris culture supernatant

Induce part1 1.png
Equipment: 100mlLB(ampicillin100μg/ml) ×4
Inoculate P. aeruginosa bacterial solution to a bottle of 100ml. NB Culture medium Incubateovernightat32℃ in a shaker 6000xg10minCentrifugal(45ml×2) 18℃cooling20-30min 200μm IPTG part1×2(each50ml) 6000xg10min cultivate4-6hours
Experiment settings: 4samples: — no supernatant+no IPTG — no supernatant+IPTG — LB+IPTG — NB+IPTG
Experiment process: Configural at 6000xg for 10min×4 bacterial liquid 1ml miliQ water resuspension. —no supernatant+no IPTG(C1C2C3) —no supernatant+IPTG(D1D2D3) —LB+IPTG(F1F2F3) —NB+IPTG(E1E2E3) Use an Absorbance Reader (562nm detection wavelength) to view fluorescence value
Induce part1 2.png
Induce part1 3.png

2. Detect red fluorescent protein production by induced detection type part1 bacteria

Experimental purpose: Draw a standard curve with different concentrations of the configured standard AHL Experimental equipment: microplate reader, fluorescence microscope
Experimental process:
(1) Inoculate the part1 bacterial solution of the bacterium to 200ml LB medium containing 100μg/ml ampicillin (200ml LB liquid culture Add 400μl 50mg/ml ampicillin stock solution to the base, and incubate overnight at 32°C on a shaker.
(2) After pre-cooling the bacterial solution at 18°C for 20-30 minutes, add isopropyl thiogalactoside (IPTG) with a final concentration of 200μM. Induce at 25°C for 6h (add 80μl 0.5mM IPTG stock to 200ml bacterial solution).
(3) Take out 11 tubes of 200ml bacterial solution, separate 15ml of each tube, 6000xg, 10min, remove the supernatant.
(4) Use 500 ul of 10 LB medium with different concentrations of AHL to suspend the bacteria in each tube (pay attention to mark), the 11th tube is only used Resuspend the bacteria in 500ul LB medium (as a control) [First prepare 1mg/ml AHL solution, weigh 1mg AHL powder and dissolve it to 1ml In LB medium, a 1:1 gradient dilution method was used to obtain a total of 10 LB mediums with different AHL concentrations]
(5) Continue to incubate at 32°C for 5 hours (slightly loosen the cap of the separation tube), proceed to separation, 6000xg, 10 minutes, and remove the supernatant.
(6) Each tube of bacteria was resuspended with 300ul miliQ water and added to 3 wells, 100ul was added to one well, a total of 33 wells (record each well the location of the inoculum).
(7) Use a microplate reader to detect (red fluorescent protein uses 562nm detection wave), after the detection is completed, export the data and save it.
(8) Temporary glass was made with the part1 bacterial liquid of the process bacteria, and the fluorescent microscope was used to observe and take photos.
Making slides:
Induced detection 1.png
Experimental data:
Different concentrations in 10 tubes after serial dilution: 1. 1 mg/ml 2. 0.5 mg/ml 3. 0.25 mg/ml 4. 0.125 mg/ml 5. 0.0625 mg/ml 6. 0.03125 mg/ml 7. 0.015625 mg/ml 8. 0.0078125 mg/ml 9. 0.00390625 mg/ml 10. 0.001953125 mg/ml Places of 12 tubes on microplate: 1. a1, 2, 3 2. b1, 2, 3 3. c1, 2, 3 4. d1, 2, 3 5. e1, 2, 3 6. f1, 2, 3 7. g1, 2, 3 8. h1, 2, 3 9. a10, 11, 12 10. b10, 11, 12 11. c10, 11, 12 12. d10, 11, 12 Using different concentrations of AHL to induce the production of red fluorescent proteins of the detectable part1 bacteria microplate reader data:
Induced detection 2.png
Images under fluorescent microscope with 12 tubes (part of):
Induced detection 3.png Induced detection 4.png Induced detection 5.png

To affirm that the plasmid is effective, we transformed it into E. coli BL21, which is a competent cell. E. bsuahlscout was induced by IPTG to express LuxR protein, and was mixed with P. aeruginosa supernatant, which made it red, and use fluorescence microscope to observe it. After we confirmed that E. bsuahlscout is effective, we used N-Acyl Homoserine Lactone (AHL) of different concentration to induce E. bsuahlscout to generate red fluorescence protein, so as to draw the standard curve of AHL concentration versus red fluorescence intensity.

3. Verify the response of part1 engineering bacteria to different concentrations of AHL

Experiment process:
After pre-cooling the bacterial solution at 18°C for 20-30 minutes, add 80μL of 0.5mM IPTG stock solution
Induced at 25℃ for 6h
Take out 12 tubes of 200ml bacterial liquid
Verify the response of part1 1.png
Centrifuge each tube with 15ml, 6000xg, 10min, remove the supernatant
Add 0.9mg of AHL powder to the first tube, 100μL of DMSO to dissolve the powder, and then add 800μL of LB medium
(DMSO: preservation of bacterial liquid)
Add 450μL of LB medium to the remaining tubes
Take 450μL from the first tube and add it to the second tube
And so, on
Verify the response of part1 2.png
Add only LB medium to the 11th tube
Add 100μL DMSO to the 12th tube and then 800μL LB medium
Resuspend the bacteria after adding LB medium containing different AHL concentrations
Verify the response of part1 3.png
Open the cap, use a breathing film to seal the isolation tube, and then use a sealing film to seal
32℃ overnight cultures.
Verify the response of part1 4.png

We sent our E. bsuahlscout to team Jilin_China to test the validity of this construct for the detection of the AHL concentration in the environment.The result showed that the E. bsuahlscout successfully expressed mCherry to change the color to pink in high AHL concentration.Since the quorum sensing signaling molecules of the Gram-negative bacteria (AHL) have various types, we realize that the types of AHL in Jilin_China’s lab and ours are different. Specifically, AHL in Jilin_China’s lab is N-(β-Ketocaproyl)-DL-homoserine lactone while ours is N-(3-Oxododecanoyl)-L-homoserine lactone. Therefore, we carefully convert the formula weights of the two types of AHL into the same concentration.Eventually, Jilin_China modified the protocol and conducted experiments that showed the more visible color expression by E. bsuahlscout in low AHL concentration.

After centrifugation. The more conspicuous pink color expression of E. bsuahlscout of the experiment by the modified protocol (Left)The more conspicuous pink color expression of E. bsuahlscout of a repeating experiment by the modified protocol(Right).

  • Partnership result.png

    Characterization by BS_United_China 2022

    We decided to utilize the LURX-AHL system for a safety system in our project. The quorum sensing suicidal system consists of three parts: LUXR, CRISPRi/dCas9, and sgRNA. The LUXR protein is regulated by the T7 promoter, lac operator, and T7 terminator. CRISPRi/dCas9 is regulated by a quorum sensing promoter and quorum sensing terminator. sgRNA is regulated by the H1 promoter and H1 terminator. After AHL is detected, the system, led by sgRNA, will target the ATP synthase of E.coli, killing the potential risk of contamination to the natural environment.

    BS-united China has improved the system by using the QS system to express the suicide gene expression.

    Sequence and Features

    Assembly Compatibility:
    • 10
    • 12
    • 21
    • 23
    • 25
    • 1000

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