Difference between revisions of "Part:BBa K855004"
 
Line 40: Line 40:
 
<p>1.Clevenger K D, Wu R, Er J A V, et al., Rational Design of a Transition State Analogue with Picomolar Affinity for Pseudomonas aeruginosa PvdQ, a Siderophore Biosynthetic Enzyme[J]. Acs Chemical Biology, 2013, 8(10):2192-2200.</p>
 
<p>1.Clevenger K D, Wu R, Er J A V, et al., Rational Design of a Transition State Analogue with Picomolar Affinity for Pseudomonas aeruginosa PvdQ, a Siderophore Biosynthetic Enzyme[J]. Acs Chemical Biology, 2013, 8(10):2192-2200.</p>
 
<p>2. Liu Y, Ebalunode J O, Briggs J M, Insights into the substrate binding specificity of quorum-quenching acylase PvdQ[J]. Journal of Molecular Graphics and Modelling, 2019.</p>
 
<p>2. Liu Y, Ebalunode J O, Briggs J M, Insights into the substrate binding specificity of quorum-quenching acylase PvdQ[J]. Journal of Molecular Graphics and Modelling, 2019.</p>
 +
===Protocols===
 +
<p>Combined with the results of experiments this year, we propose a new procedure to characterize the enzyme activity.</p>
 +
<p>1. Cut the plasmid with single enzyme</p>
 +
<p>2. Use Gibson assembly kit to ligate it with the target gene (homologous arms and His tag were added when the genes were synthesized)</p>
 +
<p>3. Transform the ligation products into E.coli BL21 and culture it after spread plate</p>
 +
<p>4. Pick some single colonies into fresh medium</p>
 +
<p>5. Use colony PCR to find the positive ones, and send samples to company to do gene sequencing</p>
 +
<p>6. Amplify the bacteria in larger volume of fresh medium</p>
 +
<p>7. Use IPTG to induce the expression of the enzymes</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>
 +
<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>(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> (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>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>12. The same operation can be done to attain different Km values towards 3-oxo-C12-HSL and C4-HSL in Pseudomonas aeruginosa.</p>
 +
<p>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</p>
 +
<p>14. Decide whether the enzyme is suitable for our project
 +
 +
</p>

Latest revision as of 16:08, 27 October 2020


pvdQ gene form pseudomonas aeruginosa PAO1 with his tag added before stop condon

pvdQ gene codes for an acylase that can degrade long-chain Nacylhomoserine lactones (AHLs), e.g. 3-oxo-C12-HSL.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1489
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1489
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1196
    Illegal XhoI site found at 1594
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1489
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1489
    Illegal NgoMIV site found at 31
    Illegal NgoMIV site found at 770
    Illegal NgoMIV site found at 1172
    Illegal NgoMIV site found at 1329
    Illegal NgoMIV site found at 1556
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1926


Kinetic Parameters and Enzyme Activity in different conditions: WHU-China 2020

BBa_K855004 parameters

Fig1: Steady-State Rate Constants for PvdQ-Catalyzed Hydrolysis of Selected Substrates. a:not determined because limited substrate solubility prevents determination of Vmax. b:Fitting error is ≤8%.
Fig2: Distribution of the various interaction energies of PvdQ-Ligand complexes.

The Michaelis constant (Km) and kcat value of an enzyme have always been important as they show the efficiency of the enzyme towards its substrate. So they are frequently used to decide whether the enzyme is “good” or not according to specific situation. And the various energy changes may also provide an insight of the enzyme-ligand interactions. So we submit the data we obtain from literatures[1][2] about pvdQ gene, including kinetic parameters towards different substrates and energy changes in the enzyme-ligand interaction process. Further users can conduct their experiments or models based on these data then.

Reference:

1.Clevenger K D, Wu R, Er J A V, et al., Rational Design of a Transition State Analogue with Picomolar Affinity for Pseudomonas aeruginosa PvdQ, a Siderophore Biosynthetic Enzyme[J]. Acs Chemical Biology, 2013, 8(10):2192-2200.

2. Liu Y, Ebalunode J O, Briggs J M, Insights into the substrate binding specificity of quorum-quenching acylase PvdQ[J]. Journal of Molecular Graphics and Modelling, 2019.

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