Part:BBa_K4817013
MomL: AHL lactonase
MomL is an AHL lactonase from Muricauda olearia Th120, belonging to the class Flavobacteriia. It can degrade both short- and long-chain AHLs. Liquid chromatography-mass spectrometry analysis demonstrated that MomL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. It contains an N-terminal signal peptide, which may affect whether it can be secreted out of warranty and affect its function of degradation of AHLs.
| momL | |
| Function | AHLs-degradation |
| Use in | Prokaryotes |
| Backbone | pET-28a |
| Derived from | Muricauda olearia Th120 |
Design and Properties:
The coding sequences of MomL and Del-MomL were connected to LacO/LacI (BBa_K1624002, BBa_K3257045) and pT7 (BBa_K4609008). IPTG was used to induce protein expression, simulating quorum sensing-induced protein expression to verify the function efficiency of the MomL. LacO/LacI are commonly found in the pET plasmids. IPTG (isopropyl β-D-1-thiogalactopyranoside) is a molecular analogue of allolactose and has the same function as allolactose. Both can act as inducers and bind to the repressor in the Lac operon, thereby preventing LacI from binding to LacO upstream of pT7 and ultimately initiating the expression of MomL. MomL crude extract was collected and used to treat E.coli DH5α(with p15A-lux-sfGFP). Without MomL, the LuxR secreted by J23100 in plasmid p15A (BBa_C0062) interacts with AHLs and initiates LuxP, expressing the green fluorescent protein sfGFP to emit fluorescence. When MomL exists, MomL degrades AHLs, LuxP cannot turn on the expression of sfGFP, and the green fluorescence weakens.
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Figure.3 pET-28a-MomL-C-His(Left) and pET-28a-Del-MomL-C-His(Right)
Figure.4 The inhibitory effect of MomL and Del-MomL on SRB biofilm (7h)
Crystal violet staining results showed that MomL and Del-MomL had inhibitory effect on the biofilm of SRB, and the effect of Del-MomL was more obvious.
Experimental approach:
1. Express and extract proteins
(1) Transform pET-28a-MomL-C-His and pET-28a-Del-MomL-C-His into DH5α strain（K+）
(2) Pick a single colony and culture it in K+ LB liquid medium overnight at 37°C and 220rpm;
(3) Extract the plasmid, transform the plasmid into BL21 (DE3), sequence the normal bacteria, and culture it in K+ LB liquid medium at 37℃ and 220rmp overnight;
(4) Take 1mL bacterial liquid cultured overnight and add it to 50 ml (250 ml Erlenmeyer flask) of K+ LB liquid culture medium, and expand the culture medium at 37°C , 220 rpm for 4 hours until OD600 =0.6-0.8;
(5) Take 10mL of bacterial liquid and store it at 4℃ for later use. Freeze 40mL of bacterial liquid at 4℃ for 5 minutes and then add IPTG (working concentration is 1mmol/L) and induce at 28℃, 200rmp for 12h;
(6) Adjust OD600 of the induced bacterial liquid to approximately the same value. Take 10mL of the induced bacterial liquid and store it at 4°C;
(7) Take 30 mL of the induced bacterial liquid, centrifuge it at 8000 rpm, 4°C for 10 min, and take 1mL of the supernatant for SDS-PAGE verification;
(8) Resuspend the pellet in 5ml 1x PBS, centrifuge at 8000rpm, 4°C for 10 minutes;
(9) Resuspend the pellet in 4ml of bacterial protein preparation lysate(with Tris-HCl) Add 1uL DNase/RNase; dispense into 2mL centrifuge tubes; incubate at 37°C, 600rpm for 30 minutes;
(10) 30% Ultrasonic power, lyse for 10 seconds, rest for 10 seconds, a total of 10 minutes; 5 minutes interval, repeat 2-3 times;
(11) Centrifuge at 13000g, 4°C for 30 minutes, take the supernatant as crude protein solution, and store it at -20°C;
(12) In a clean bench, filter the crude protein solution with a 0.45μm filter membrane to sterilize.
2. 96-well microtiter plate assay (Crystal violet staining of biofilms)[2]
(1) Incubate the bacterial solution overnight for 12 hours until the OD600>1. Add antibiotic-free LB dilution at a ratio of 1:10. Add 125µl of the diluted bacterial solution to each well of a 96-well plate. Inoculate and incubate overnight at 37°C without shaking for 24 hours. (LB-only medium is required as a control)
[SRB bacterial film needs to be cultured in an anaerobic bag for 7 hours]
(2) Aspirate the LB, add 150ul of lysis supernatant containing induced expression protein (sterilized), and place at a constant temperature of 37℃ Celsius for 18 hours.
[Wash the 96-well plate with biofilm twice with 200ul of sterile water, add 100ul of lysis solution, scrape off the film with a pipette tip, and spread it on the plate. After 12 hours, observe the number of single colonies growing on the plate.]
[It is also feasible to directly stain and observe the growth of a certain type of bacterial film without adding lysis solution.]
(3) Aspirate the liquid, gently soak the well plate in 1L of distilled water and wash it twice. When the plate is submerged, gently wipe the surface of the plate with gloved fingers to release air bubbles and ensure that water enters. Turn the plate up side down and tap hard. Place the 96-well plate upside down on absorbent paper to remove as much water as possible.
(4) Add 200μL of 0.1% crystal violet solution (containing 5% methanol) into the well. This volume ensures that the stain covers the biofilm. Let sit for 10 minutes. Invert the plate in the waste tray and shake gently to remove the liquid.
(5) Gently soak the well plate in 1L of distilled water and wash it twice, and rub the entire surface of the plate to ensure that water enters all wells. Remove the plate from the water, invert, and shake to remove liquid. Replace with distilled water and repeat the above steps twice.
(6) Turn the plate upside down and tap hard. Place the 96-well plate upside down on absorbent paper to remove as much water as possible.
(7) Place the washed 96-well plate into the oven until the water is completely dry
(8) Add 200ul of 95% ethanol to each well and wait for 10 minutes until the crystal violet is completely dissolved.
(9) Use a microplate reader to measure the OD570nm (at least 3 repeat groups)
Note：If you’d like to count the single colonies on the plate that spread the biofilm, it is better to use resistant membrane-producing strains to avoid contamination.
3. AHLs Degradation Assay
(1) Use E.coli DH5α with the p15A-LuxR-sfGFP plasmid, set a control group, and measure the fluorescence intensity to reflect the degradation effect of the enzyme on AHLs.
(2) Cultivate the newly transformed E.coli DH5α (with p15A-lux-sfGFP) overnight for about 8 hours, and adjust the OD600 to about 0.6.
(3) Add 250ul of bacterial solution to each well; add 10ul of AHLs to the AHL-treated wells; the total volume of the protein solution is 30ul.
(4) Set the microplate reader program to measure the fluorescence and OD600 of the entire 96-well plate every 10 minutes for a total of 2 hours and 11 test values. Finally, measure the fluorescence of the odd-numbered column and the OD600 of even-numbered column, and use fluorescence intensity/OD600 to evaluate the intensity of bioluminescence.
[Note: The bacterial solution should be added last to avoid inaccuracy due to differences in operating time]
4. PCR-based Site-directed Mutagenesis Method
(1) Design primers: Select the mutation point and 15bp before it, 33 bp in total, as F-primers. Select the mutation point and 15bp after it, 33 bp in total, as R-primers.
(2) PCR(25μL in total/50μL in total); After electrophoresis and gel recovery, obtain the linearized mutant plasmids.
(3) Digest the original plasmid with DpnⅠ, and then use ligase to circularize the plasmid to obtain the mutant plasmid.
Reference
[1] Tang K, Su Y, Brackman G, Cui F, Zhang Y, Shi X, Coenye T, Zhang XH. MomL, a novel marine-derived N-acyl homoserine lactonase from Muricauda olearia. Appl Environ Microbiol. 2015 Jan;81(2):774-82. doi:10.1128/AEM.02805-14.
[2] Coffey, B.M., Anderson, G.G. (2014). Biofilm Formation in the 96-Well Microtiter Plate. In: Filloux, A., Ramos, JL. (eds) Pseudomonas Methods and Protocols. Methods in Molecular Biology, vol 1149. Humana, New York, NY.
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