Difference between revisions of "Part:BBa K3562001"

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AiiO (with His-tag)

AHL-acylase with AHLs (Quorum-sensing factor Acyl Homoserine Lactone) degradation activity.

Sequence and Features

Usage and Biology

AHL-acylase is a acylase that cleaves AHL by hydrolyzing the amide linkage between the acyl side chain and HSL moiety.(Fig.1)

Figure 1 Mechanism of AHL-acylase

Quorum sensing factor is closely related to bacterial virulence. Enzymes that degrade AHL can control the concentration of AHL to silence the expression of related genes. In our project, we use these enzymes to control the virulence of Pseudomonas aeruginosa referred to existing experimental therapies.

Different AHL degrading enzymes have different substrate preferences and degradation activities. We try to select suitable ones from a variety of enzymes.

Same as other acylases, AiiO catalyzes the reaction which cleaves the amide bond of AHL and turns it into a free homoserine lactone and a fatty acid. And it locates inside the bacteria cell. As for the substrate specificity, it may have a strong preference for inactivating AHLs with the acyl side chains greater than eight carbons and may exhibit a slight preference for 3-oxo-substituted AHLs, However, it probably can degrade several short-chain AHLs as well. So it may function as a broad-spectrum acylase inside the cell.

Figure 2 Reduction of AHL concentration during incubation with Ochrobactrum sp. 44 cell extracts. a: This analysis was performed in duplicates. The mean value and the standard errors (indicated as ±value) were calculated. b: Percentage values indicate the initial concentration of AHL. c: Percentage values indicate the concentration of remaining AHL.[1]

Protocols

Combined with the results of experiments this year, we propose a new procedure to characterize the enzyme activity.

1. Cut the plasmid with single enzyme

2. Use Gibson assembly kit to ligate it with the target gene (homologous arms and His tag were added when the genes were synthesized)

3. Transform the ligation products into E.coli BL21 and culture it after spread plate

4. Pick some single colonies into fresh medium

5. Use colony PCR to find the positive ones, and send samples to company to do gene sequencing

6. Amplify the bacteria in larger volume of fresh medium

7. Use IPTG to induce the expression of the enzymes

8. Collect the bacteria and extract the proteins

9. Keep some of the crude bacteria extract and use Ni resins (from commercial kit) to purify others

For the crude extract:

(1) Mix the crude extract and the AHL standard stock solutions in certain buffer (according to the optimal PH of the enzyme) and the control group is added with extract inactivated by high temperature

(2) React in optimal temperature and take one sample per 30 minutes

(3) Extract three times with at least an equal volume of acidified ethyl acetate

(4) The organic layer is separated, collected and dried using anhydrous sodium sulfate

(5) Use a rotary evaporator to remove the organic solvent in the sample under reduced pressure

(6) The residue is reconstituted in chromatographic grade methanol

(7) The sample is analyzed by HPLC to quantify the AHL concentration in the reaction system

For the purified enzymes:

(1) Analyze the protein with SDS-PAGE

(2) Measure the enzyme concentration by BCA kit

If the crude extract can efficiently degrade AHLs, then use the purified enzymes to repeat the former procedure performed on the crude extract; if not, change the conditions of enzyme expression to avoid inclusion-body form of the enzymes

For the standard curve:

(1) Dissolve certain amount of AHL dry powder in chromatographic grade methanol to create a series of AHL solutions with different concentrations

(2) Analyze them by HPLC and draw the standard curve of AHL concentration versus HPLC data.

10. Analyze the data from the experiments of purified enzymes with the help of the standard curve

11. Then use excel to draw the Lineweaver-Burk plot and obtain the Km (Michaelis constant) value of the enzymes towards this kind of AHL

12. The same operation can be done to attain different Km values towards 3-oxo-C12-HSL and C4-HSL in Pseudomonas aeruginosa.

13. Combine the data with our quorum dynamics model to observe the overall effects the enzymes have on the quorum sensing systems of P.aeruginosa

14. Decide whether the enzyme is suitable for our project

Reference

[1]Czajkowski R, Krzyzanowska D, Karczewska J, et al., Inactivation of AHLs by Ochrobactrum sp. A44 depends on the activity of a novel class of AHL acylase[J]. Environmental Microbiology Reports, 2011, 3(1):59-68.