Granted Patent
B2
US 12,594,307 · App. 17/784,547 · Granted Apr 7, 2026
Compositions and methods of using inducible signaling for tunable dynamics in microbial communities
Inventors:
Jeff Hasty (San Diego, CA); Arianna Miano (La Jolla, CA); Michael Julius Liao (San Diego, CA)
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Abstract
Provided herein are compositions and methods for modulating induction of quorum sensing in bacterial cells. For example, provided herein is a method of inducing method of inducing quorum sensing, where the method includes: culturing a bacterial strain, wherein the bacterial strain comprises a first nucleic acid sequence encoding a first activator polypeptide, wherein expression of the first activator polypeptide produces a quorum sensing molecule precursor; a second nucleic acid sequence encoding a second activator polypeptide, wherein expression of the second activator polypeptide produces a quorum sensing; a third nucleic acid sequence encoding a third activator polypeptide that is capable of activating the quorum sensing system; a fourth nucleic acid sequence encoding a gene of interest, and contacting the bacterial strain with an inducer molecule; and converting the inducer molecule into a quorum sensing molecule, thereby allowing induction of quorum sensing.
Claims (19)
1 . A method of inducing quorum sensing, the method comprising:
(a) culturing an Escherichia coli bacterial strain, wherein the Escherichia coli bacterial strain comprises
(i) a first nucleic acid sequence encoding p-coumaric acid-CoA ligase from Nicotiana tabacum (4CL2nt), wherein expression of the p-coumaric acid-CoA ligase first activator polypeptide results in production of coumaroyl-CoA, wherein the first nucleic acid sequence is operably linked to a pLux promoter;
(ii) a second nucleic acid sequence encoding an RpaI synthase, wherein expression of the RpaI synthase results in production of p-coumaroyl-homoserine lactone from coumaroyl-CoA, wherein the second nucleic acid sequence is operably linked to a pLux promoter;
(iii) a third nucleic acid sequence encoding an RpaR transcriptional regulator, wherein expression of the RpaR transcriptional regulator results in activation of a quorum sensing system, wherein the third nucleic acid sequence is operably linked to a pLux promoter; and
(iv) a fourth nucleic acid sequence encoding a reporter protein, wherein the second and third nucleic acid sequences together form the quorum sensing system responsive to p-coumaroyl-homoserine lactone the fourth nucleic acid sequence is operably linked to the J23106 constitutive promoter; and
(b) contacting the Escherichia coli bacterial strain with p-coumaric acid; and
(c) converting p-coumaric acid into p-coumaroyl-homoserine lactone via steps (i)-(ii), thereby inducing quorum sensing.
2 . The method of claim 1 , wherein the quorum sensing system comprises the Rpa quorum sensing components RpaI synthase and RpaR transcriptional regulator obtained from Rhodopseudomonas palustris.
3 . A bacterial strain produced by the method of claim 1 .
4 . A set of nucleic acids comprising the nucleic acid sequences of claim 1 .
5 . A composition comprising the bacterial strain of claim 3 .
6 . The method of claim 1 , wherein the first nucleic acid sequence comprises an amino acid sequence with at least 90% identity to SEQ ID 1.
7 . The method of claim 1 , wherein the reporter protein is a fluorescent protein selected from the group consisting of green fluorescent protein (GFP), superfolder GFP (sfGFP), or cyan fluorescent protein (CFP).
8 . The method of claim 1 , wherein the Escherichia coli bacterial strain further comprises a fifth nucleic acid sequence encoding a bacteriophage lytic protein capable of forming a lesion in the membrane of a host cell.
9 . The method of claim 8 , wherein the bacteriophage lytic protein is from bacteriophage ΦX174.
10 . The method of claim 8 , wherein the fifth nucleic acid sequence encoding the bacteriophage lytic protein is operably linked to a promoter that is transcriptionally regulated by the RpaR transcriptional regulator.
11 . The method of claim 10 , wherein expression of the bacteriophage lytic protein is sufficient to initiate synchronized lysis of the Escherichia coli bacterial strain, resulting in cyclic population death and cargo release.
12 . The method of claim 1 , wherein the step (b) contacting the Escherichia coli bacterial strain with p-coumaric acid comprises maintaining the concentration of p-coumaric acid between about 15 μM to 1 mM.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Jun 13, 2022
From: HASTY, JEFF; MIANO, ARIANNA; LIAO, MICHAEL JULIUS
To: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Reel/Frame 060185/0144 →
Continuity (2)
Provisional Application
62947932
· Dec 13, 2019
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