High throughput in vitro translation (cell-lysate based) assay for detecting quorum sensing signals
A cell-lysate extract based assay reagent for detecting quorum sensing signals is generally provided, along with methods of making and using the same. The assay reagent generally includes a cell-lysate extract formed from a biosensor bacterium (e.g., Agrobacterium tumefaciens NTL4 (pCF218)(pCF372)) and a detecting substrate (e.g., an absorbance-based or luminescence-based substrate). The cell-lysate extract can be prepared by (1) disrupting the cell membranes of the biosensor bacterium to release the cellular components into a solution, (2) centrifuging the resulting solution, and (3) removing the resulting supernatant solution.
1. A method of making an assay reagent from a cell culture of Agrobacterium tumefaciens NTL4 (pCF218)(pCF372) for detecting a quorum sensing signal, the cell culture comprising cellular contents within a cell membrane, the method comprising:
disrupting the cell membrane of Agrobacterium tumefaciens NTL4 (pCF218)(pCF372) to release the cellular contents into an aqueous solution;
centrifuging the aqueous solution to form a supernatant containing the cellular contents;
collecting the supernatant solution to separate the supernatant solution from the cell membrane; and
adding a detecting substrate to the supernatant solution to form an assay reagent for detecting the presence of a quorum sensing signal, wherein the detecting substrate is an absorbance-based substrate or a luminescence-based substrate.
2. The method as in claim 1 , wherein disrupting the cell membrane of the biosensor bacterium to release the cellular contents comprises applying sound energy to the biosensor bacterium to sonicate the cell membranes.
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-galactopyranoside.
5. The method as in claim 1 , wherein the detecting substrate comprises a luminescence-based substrate.
6. The method as in claim 5 , wherein the luminescence-based substrate comprises 6-O-β-galactopyranosyl luciferin.
7. The method as in claim 1 , disrupting the cell membrane:
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
applying sound energy to the cells to disrupt the cell membranes and release the cellular contents.