Difference between revisions of "Part:BBa K3562001"
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AHL-acylase with AHLs (Quorum-sensing factor Acyl Homoserine Lactone) degradation activity. | AHL-acylase with AHLs (Quorum-sensing factor Acyl Homoserine Lactone) degradation activity. | ||
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<partinfo>BBa_K3562001 parameters</partinfo> | <partinfo>BBa_K3562001 parameters</partinfo> | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
<p> | <p> | ||
| − | AHL-acylase is | + | AHL-acylase is an acylase that cleaves AHL by hydrolyzing the amide linkage between the acyl side chain and HSL moiety.(Fig.1) |
</p> | </p> | ||
[[Image:AHL-acylase.png|frame|'''Figure 1''' Mechanism of AHL-acylase]] | [[Image:AHL-acylase.png|frame|'''Figure 1''' Mechanism of AHL-acylase]] | ||
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</p> | </p> | ||
[[Image:AiiO fig2.png|frame|'''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]]] | [[Image:AiiO fig2.png|frame|'''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]]] | ||
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===Protocols=== | ===Protocols=== | ||
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<p>7. Use IPTG to induce the expression of the enzymes</p> | <p>7. Use IPTG to induce the expression of the enzymes</p> | ||
<p>8. Collect the bacteria and extract the proteins</p> | <p>8. Collect the bacteria and extract the proteins</p> | ||
| − | <p>9. Keep some of the crude bacteria extract and use Ni resins (from commercial kit) to purify others | + | <p>9. Keep some of the crude bacteria extract and use Ni resins (from commercial kit) to purify others</p> |
<p>For the crude extract:</p> | <p>For the crude extract:</p> | ||
| − | + | <p>(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</p> | |
| − | + | <p>(2) React in optimal temperature and take one sample per 30 minutes</p> | |
| − | + | <p>(3) Extract three times with at least an equal volume of acidified ethyl acetate</p> | |
| − | + | <p>(4) The organic layer is separated, collected and dried using anhydrous sodium sulfate</p> | |
| − | + | <p>(5) Use a rotary evaporator to remove the organic solvent in the sample under reduced pressure</p> | |
| − | + | <p>(6) The residue is reconstituted in chromatographic grade methanol</p> | |
| − | + | <p>(7) The sample is analyzed by HPLC to quantify the AHL concentration in the reaction system</p> | |
<p>For the purified enzymes:</p> | <p>For the purified enzymes:</p> | ||
| − | + | <p>(1) Analyze the protein with SDS-PAGE</p> | |
| − | + | <p>(2) Measure the enzyme concentration by BCA kit</p> | |
| − | + | <p>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</p> | |
<p>For the standard curve:</p> | <p>For the standard curve:</p> | ||
| − | + | <p> (1) Dissolve certain amount of AHL dry powder in chromatographic grade methanol to create a series of AHL solutions with different concentrations</p> | |
| − | + | <p>(2) Analyze them by HPLC and draw the standard curve of AHL concentration versus HPLC data.</p> | |
<p>10. Analyze the data from the experiments of purified enzymes with the help of the standard curve</p> | <p>10. Analyze the data from the experiments of purified enzymes with the help of the standard curve</p> | ||
<p>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</p> | <p>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</p> | ||
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===Reference=== | ===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. | [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. | ||
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| + | <span class='h3bb'>Sequence and Features</span> | ||
| + | <partinfo>BBa_K3562001 SequenceAndFeatures</partinfo> | ||
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| + | <!-- Uncomment this to enable Functional Parameter display | ||
| + | ===Functional Parameters=== | ||
| + | <partinfo>BBa_K3562001 parameters</partinfo> | ||
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Latest revision as of 11:52, 18 October 2021
AiiO (with His-tag)
AHL-acylase with AHLs (Quorum-sensing factor Acyl Homoserine Lactone) degradation activity.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]