High-throughput in vitro translation (cell-lysate based) assay for detecting quorum sensing signals

1. A method of detecting a quorum sensing signal in a bacteria sample, the method comprising:

adding a detecting substrate and the cell-lysate extract solution to a well of a microtiter plate to form an assay reagent configured to report the detection of the quorum sensing signal, wherein the detecting substrate is an absorbance-based substrate or a luminescence-based substrate, and wherein the cell-lysate extract solution is formed from Agrobacterium tumefaciens NTL4 (pCF218)(pCF372);

adding the bacteria sample to the well;

incubating the bacteria sample within the well in the presence of the cell-lysate extract solution;

measuring absorbance or luminescence of the assay reagent in the presence of the bacteria sample within the well; and

determining the presence or absence of quorum sensing signals in the bacteria sample, wherein the quorum sensing signal comprises an acyl-homoserine lactone.

2. The method as in claim 1 , wherein the assay reagent is an aqueous-based solution comprising the detectable substrate and the cell-lysate extract solution.

3. The method as in claim 1 , wherein the detecting substrate comprises an absorbance-based substrate.

4. The method as in claim 3 , wherein the absorbance-based substrate comprises 5-bromo-4-chloro-3-indolyl-β-D-galactopyransoside.

5. The method as in claim 3 , wherein absorbance is measured by:

applying a light having a specific wavelength to the assay reagent in the presence of the bacteria sample, and

measuring any absorbance of the sample at the specific wavelength.

6. The method as in claim 1 , wherein the detecting substrate comprises a luminescence-based substrate.

7. The method as in claim 6 , wherein the luminescence-based substrate comprises 6-O-β-galactopyranosyl luciferin.

8. The method as in claim 6 , wherein luminescence is measured by reading the luminescence of the sample using a luminometer.

9. The method as in claim 1 , wherein forming the cell-lysate extract solution from the biosensor bacterium comprises:

centrifuging a cell culture to create cell pellets;

collecting the cell pellets comprising cells, wherein each cell comprises cellular contents surrounded by a cell membrane; and

disrupting the cell membranes of the cells to release the cellular contents into an aqueous solution.

10. The method as in claim 9 , wherein disrupting the cell membranes of the cells to release the cellular contents into the solution comprises:

applying sound energy to the cells to disrupt the cell membranes and release the cellular contents.

11. The method as in claim 9 , further comprising:

centrifuging the aqueous solution to form the cell-lysate extract solution as a supernatant solution containing the cell contents separated from the cell wall membranes.

12. The method as in claim 1 , wherein the bacteria sample is incubated with the well prior to adding the detecting substrate and the cell-lysate extract solution.

13. The method as in claim 12 , further comprising:

after adding the detecting substrate and the cell-lysate extract solution to the well, incubating the detecting substrate with the cell-lysate extract solution.

14. The method as in claim 1 , wherein the detecting substrate and the cell-lysate extract solution are added after the bacteria sample is added to the well.

15. The method as in claim 2 , wherein the assay reagent is a buffered aqueous-based solution comprising a buffering agent and having a pH that is balanced from about 5 to about 9.

16. The method as in claim 15 , wherein the buffered aqueous-based solution has a pH that is balanced from about 6 to about 8.

17. The method as in claim 15 , wherein the buffered aqueous-based solution comprises KH 2 PO 4 .