Difference between revisions of "Part:BBa C0160"

Revision as of 16:05, 27 October 2020

autoinducer inactivation enzyme aiiA (no LVA)

same as Part:BBa_C0060 except no LVA tag

Sequence and Features

Characterization done by ETH Zurich 2015

Check the characterization done by ETH Zurich 2015 iGEM team in the experience page.

Functional Parameters: Austin_UTexas

BBa_C0160 parameters

Burden Imposed by this Part:

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.

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

BBa_C0160 parameters

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 what is also important is the conditions influencing the enzyme activity, such as temperature, PH, metal ions and so on. Here we add the data we obtain from literature[1] about aiiA gene, including kinetic parameters towards different substrates and its activity changes versus different conditions. Further users can conduct their experiments or models based on these data then.

Reference:

1.Wang L H, Weng L X, et al., Specificity and Enzyme Kinetics of the Quorum-quenching N-Acyl Homoserine Lactone Lactonase (AHL-lactonase) [J]. Journal of Biological Chemistry, 2004, 279(14):13645-51.

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