Part:BBa_K4817012
DisH
DisH is the dispersal hexosaminidase for DVU2239, an N-acetyl β-D-hexosaminidase secreted by a member of the SRB family namedDesulfovibrio vulgaris. It is a glycoside hydrolase from family 3 domain protein [D. vulgaris DP4][1]and can disperse biofilms secreted by D. vulgaris. It mainly acts on N-acetyl-b-D-galactosamine (GalNAc) in biological membranes. According to research[1], DisH can disperse biofilms of Pseudomonas aeruginosa, Escherichia coli and Bacillus subtilis. Purified DisH can disperse about 63% of the biofilm under optimal conditions, inhibiting biofilm formation by up to 47%, so we attempted to characterize this protein in engineered bacteria to degrade SRB biofilms.
Biology and Usage
| DisH | |
| Function | Biofilm dispersal |
| Use in | Prokaryotes |
| Backbone | pET-28a |
| Derived from | Desulfovibrio vulgaris |
Design and Properties
The coding sequence of DisH was 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 degradation efficiency of the DisH protein on the membrane. LacO/LacI are commonly found in the pET plasmids. IPTG (isopropyl β-D-1-thiogalactopyranoside) is a molecular analogue of allolactose and cannot be metabolized by common laboratory chassis such as E. coli. IPTG 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 DisH.
Figure 1. genetic circuit diagram
Figure 2. pET-28a-DisH-C-His plasmid
Figure.3 The degradation effect of DisH on different biofilms [A:The degradation effect of DisH on DH5α biofilm(24h), B: The degradation effect of DisH on BL21（DE3）-pET28a biofilm(24h), C: The degradation effect of DisH on SRB biofilm(7h)]
DiSH has good degradation effects on the biofilms of E.coli BL21(DE3), E.coli DH5α and SRB
Figure.4 Effect of BL21(DE3)-DisH on biofilm formation
The engineered bacterial biofilms were collected and plated. The number of attached bacteria to the engineered bacterial biofilm containing the plasmid pET-28a-DisH was minimal, indicating that DisH can inhibit biofilm formation.
Figure.5 α-GC Activity Assay results of DisH（left：p-nitrophenol concentration(nmol/mL) assay; right: GC activity）
The enzyme activity of the crude protein extract was measured, and the results showed that the enzyme activity of DisH was low.
Experimental approach:
1. Express and extract proteins
(1) Transform pET-28a-DisH-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.]
[It is better to use resistant membrane-producing strains to avoid contamination.]
(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) Pipette 200 μL 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 OD570 (at least 3 repeat groups)
3. α-GC Activity Assay
Follow the protocol of α-GC Activity Assay Kit and set up a control group to detect enzyme activity.
References
[1] Zhu L, Poosarla VG, Song S, Wood TL, Miller DS, Yin B, Wood TK. Glycoside hydrolase DisH from Desulfovibrio vulgaris degrades the N-acetylgalactosamine component of diverse biofilms. Environ Microbiol. 2018 Jun;20(6):2026-2037.
[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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