Difference between revisions of "Part:BBa K3882001"

Latest revision as of 11:22, 21 October 2021

E. bsuahlscout-QS-bsuahlteminator

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.

Our E. bsuahlterminator bio-brick contains 2 key elements including pvdq and eGFP. PVDQ is a protein coded by pvdq gene. The pvdq gene is regulated by T7 promoter, Lac operator and T7 terminator. The PVDQ protein can catalyze the deacylation of acyl-homoserine lactone (AHL) which can prevent the activation of LUXR by eliminating the AHL in the environment. In order to increase the stability and show the production quality in a real time manner. We use a protein linker and 3x HA tag to link the PVDQ protein with eGFP. We also use 3xHis tag at the c-terminal of eGFP to increase the efficiency to purify the recombination proteins. Our results shows the great stability in solutions and almost no aggregation happens after elusion.

Using QS promoter to start the E. bsuahlterminator bio-brick, we are able to detecting the AHL in the environment and do the clearance at the same time.

Experiment results of Part BBa K3882000:E.bsuahlscout

1. Induce part1 by using the verdigris culture supernatant

Equipment: 100mlLB(ampicillin100μg/ml) ×4
Inoculate Pseudomonas 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

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:

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:

Images under fluorescent microscope with 12 tubes (part of):

Experiment results of Part BBa K3882002: E. bsuahlterminator

1. Learn to configure IPTG and use IPTG to induce part2 cells to express green fluorescent protein.

Experimental method:
(1) Inoculate the part2 of engineered bacteria into 100ml LB medium containing 100μg/ml ampicillin (100ml LB liquid culture Add 200μl of 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 IPTG with a final concentration of 200μM (into 100ml bacterial solution, add 40μl 0.5mM IPTG stock solution), induced at 25°C for 6h. A total of 2 samples are prepared: without IPTG induction, with IPTG induction
(3) Take 15ml each of the 2 bottles of engineering bacteria part2 and centrifuge at 6000xg for 10min, then remove the supernatant.
(4) 2 tubes of bacteria were resuspended with 1mL mili-Q water each, and added to 3 wells, 100ul was added to one well for a total of 6 wells.
Note: Adding IPTG is A1, A2, A3. No IPTG is B1, B2, B3.
(5) Use the microplate reader to detect (wavelength 492nm), after the detection, export the data and save it.
(6) Use a fluorescence microscope to observe 2 bottles of engineering bacteria part2 and take photos.
Microplate reader data:


Fluorescence microscope photo (7.26):

The LUXR-AHL complex can bind to the QS promoter1 in BBa_K3882000 to express the mCherry gene. Moreover, the QS promoter 2 which is the upstream of BBa_K3882002 can be activated. The results show the expression of PVDQ-GFP.

2. Learn to configure IPTG and use IPTG to induce part2 cells to express green fluorescent protein.

Sequence and Features