Part:BBa_K4817015
AidH homoserine lactonase
AidH is a novel AHL-degrading enzyme from Ochrobactrum discovered by Gui-Ying Mei et al. [1], which can hydrolyze the lactone ring of AHL to produce acyl homoserine. However, AidH shares high similarities with members of the α/β-hydrolase fold family, which have multiple catalytic functions. It is proved that AidH has a catalytic effect on a variety of AHLs, and it is expected that the enzyme can be evolved to optimize its function and become a broader-spectrum AHL-lactonase.
In this experiment, we used the IPTG induction system and T7 promoter (pT7, BBa_K4609008) to characterize proteins under induction conditions and conduct functional verification. At the same time, we analyzed the structure of AidH and tried to optimize its structure.
We found that the side chain of the 147th alanine residue of AidH may hinder the binding of the substrate to the active pocket. [2] Therefore, we mutated alanine for glycine at amino acid 147 (AidH A147G) to reduce steric hindrance and alanine for valine(AidH A147V)to increase steric hindrance.
Biology and Usage
| AidH | |
| Function | AHLs-degradation |
| Use in | Prokaryotes |
| Backbone | pET-28a |
| Derived from | Ochrobactrum |
Design and Properties:
We linked the coding sequences of AidH and its mutants AidH A147G/AidH A147V 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 of the AidH and AidH A147G/AidH A147V. LacO/LacI are commonly found in the pET series 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 AidH.
AidH crude extract, AidH A147G crude extract, AidH A147V crude extract were collected and used to treat E.coli DH5α(with p15A-lux-sfGFP). Without AidH, 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 AidH exists, AidH degrades AHLs, LuxP cannot turn on the expression of sfGFP, and the green fluorescence weakens.
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The formation of microbial biofilm depends on the quorum sensing molecules AHLs, so the crystal violet staining results of the bacterial membrane treated with the protein solution can reflect the degradation effect of the protein on AHLs. The results of crystal violet staining show that AidH and AidH A147G/AidH A147V have good degradation effects on the biofilm of SRB. It shows that these proteins can quench quorum sensing molecules and thereby block biofilm formation.
For protein mutants, since the side chain of the 147th alanine residue of AidH may hinder the binding of the substrate to the active pocket, the degradation effect of AidH A147V with smaller steric hindrance is greater than that of AidH(WT). On the contrary, the degradation effect of AidH A147G, which is more sterically hindered, is worse than that of AidH(WT). The experimental results are consistent with the theoretical results.
Figure.5 Degradation effect of AidH crude extract at different concentrations on oxo-C6-HSL
4mL of AidH protein solution extracted from 30ml of bacterial solution, at dilution factors of 30ul/10ul/1ul (30ul in total), has a similar degradation effect on oxo-C6-HSL .
Figure.6 Degradation effect of AidH crude extract on different AHLs
Measure the AHL concentration with E.coli DH5α (with p15A-lux-sfGFP) , and reflect the AHL concentration by fluorescence intensity.
4mL of AidH/ AidH A147G/AidH A147V protein solution extracted from 30ml of bacterial solution, at dilution factors of 30ul/10ul/1ul (30ul in total), has a similar degradation effect on oxo-C6-HSL . For different AHLs, after adding 30ul of undiluted enzyme, the fluorescence intensity of all treated experimental groups was lower than that of the untreated experimental group. In general, the fluorescence intensity decreased overall.
Experimental approach:
1. Express and extract proteins
(1) Transform pET-28a-AidH-C-His, pET-28a-AidH A147V-C-His,
pET-28a-AidH A147G-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)[3]
(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.
References
[1] Mei GY, Yan XX, Turak A, Luo ZQ, Zhang LQ. AidH, an alpha/beta-hydrolase fold family member from an Ochrobactrum sp. strain, is a novel N-acylhomoserine lactonase. Appl Environ Microbiol. 2010;76(15):4933-4942.
[2] Zhang Y, Wei W, Wen H, Cheng Z, Mi Z, Zhang J, Liu X, Fan X. Targeting Multidrug-Recalcitrant Pseudomonas aeruginosa Biofilms: Combined-Enzyme Treatment Enhances Antibiotic Efficacy. Antimicrob Agents Chemother. 2023 Jan 24;67(1):e0135822.
[3] 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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