DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status Summary
Claims 1- 6, 12-16, 19-21,23-25 and 27-29 are pending.
Claims 12-16, 19-21,23-25 and 27-29 are withdrawn.
Claims 1- 6 are considered on the merits.
Claims 1- 6 are rejected.
No Claims are allowed.
Election/Restrictions
Applicant’s election without traverse of Group I, claims 1-6, without traverse in the reply filed on 2026 June 17 is acknowledged.
Claims 12-16, 19-21,23-25 and 27-29 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 2026 June 17.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1 is/are rejected under 35 U.S.C. 103 as being unpatentable over Fernandez (Fernandez et al. Proc Natl Acad Sci U S A. 2020 Nov 17;117(46):29046-29054.), and further in view of Pitner (Pitner et al. ACS Synth Biol. 2015 Sep 18;4(9):966-74.), Narasaki (Narasaki et al. J Biol Chem. 2005 Apr 8;280(14):14278-87.), and Taton (Taton et al. ACS Synth Biol. 2017 Dec 15;6(12):2175-2182.).
Claim Interpretation
Claim 1 is essentially a method comprising a dual “NOT gate” gene circuit with orthogonal promoters; the first NOT gate is defined in clause (i) of the claim and the second NOT gate is defined in clause (ii). The input (repressor) inverts expression of the biofilm and protease respectively. Similarly, the absence of the first input (biofilm repressor) activates biofilm growth, whereas absence of the second input (protease repressor) activates planktonic growth. Claims 2- 6 are similarly methods comprising gene circuits.
Fernandez realized bacteria’s shape is directly related to their transition between planktonic growth phase and biofilm growth phase. Fernandez uses a combination of genetic circuits to manipulate the shape of bacteria and in the process exhibits a “method of controlling” bacteria’s “transition between planktonic growth phase and biofilm growth phase.”
The following passage highlighting Fernandez’s ability to use wild type bacteria and engineer mutant bacteria exemplifies their “method of controlling” the bacteria’s shape and planktonic/biofilm transition, “During microcolony formation, wild-type V. cholerae cells tended to exist as straight rods, while genetically engineering cells to maintain high curvature reduced microcolony formation and biofilm density. Conversely, straight V. cholerae mutants have reduced swimming speed when using flagellar motility in liquid [medium]. Our results demonstrate regulation of cell shape in bacteria is a mechanism to increase fitness in planktonic and biofilm lifestyles.” (Abstract). Furthermore, the preceding statement exemplifies Fernandez’s method occurs in medium. Further clarification regarding “growing a bacterial host cell in a medium” is described in their material and method section Analysis of Curvature of Adhered Cells in Microcolonies and Nonadhered in Solution. Fernadez teaches “At the given time point, the media for two biological replicates were removed by aspiration…” (page 29052, 2nd column, 1st paragraph).
Fernandez further teaches their use of gated gene circuits, clauses (i) and (ii), by teaching promoters operably linked to proteins. They teach a gene circuit/clause (i), “we generated a transcriptional reporter of the crvA promoter (PcrvA) … fused to luciferase [reporter]”(page 29048, 2nd column, 2nd paragraph). The preceding statement exemplifies Fernandez’s system’s ability to incorporate the particular “recombinant polynucleotide encoding a biofilm assembly protein operably linked to a first repressible promoter” recited in the clause, where in Fernandez’s system, the luciferase would represent the biofilm assembly protein and the crvA promoter would represent the first repressible promoter. Fernandez further teaches a gene circuit/clause (ii), “We tested this hypothesis by expressing CrvA from a multicopy plasmid using the PBAD promoter (pCrvA)” (page 29049, 1st column, 3rd paragraph). The preceding statement exemplifies Fernandez’s system’s ability to incorporate the particular “recombinant polynucleotide encoding a protease capable of breaking down the one or more biofilm assembly proteins operably linked to a second repressible promoter” recited in the clause, where in Fernandez’s system, the CrvA would represent the protease protein and the PBAD promoter would represent the second repressible promoter.
Fernandez does not teach (i) … a first repressible promoter; and
(ii) … a protease capable of breaking down the one or more biofilm assembly proteins … a second repressible promoter;
wherein addition of a repressor for the first repressible promoter to the medium results in suppression of the expression of the recombinant polynucleotide encoding one or more biofilm assembly proteins and expression of the recombinant polynucleotide encoding a protease such that the bacterial host cell exhibits planktonic growth phase; and wherein the absence of the repressor for the first repressible promoter and the presence of the repressor for the second repressible promoter in the medium results in expression of the recombinant polynucleotide encoding one or more biofilm assembly proteins and suppression of the expression of the recombinant polynucleotide encoding a protease such that the bacterial host cell exhibits biofilm growth phase.
Pitner teaches gene circuits with dual orthogonal promoters where the protease is operably linked to promoter “and TEV was expressed in an arabinose-inducible fashion from the pBAD promoter” (page 967, 2nd column, 2nd paragraph; see image below). Furthermore, Pitner shows they are in the same field of endeavor and demonstrates a teaching/motivation to modify their PASS platform “PASS presents a new strategy for engineering microbial gene regulation to achieve the desirable performance characteristics exhibited by natural mechanisms while also being amenable to modular protein and gene circuit engineering.” (page 972, 1st column, bottom).
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Pitner does not teach (i) … a first repressible promoter; and
(ii) … a [protein] capable of breaking down the one or more biofilm assembly proteins … a second repressible promoter;
wherein addition of a repressor for the first repressible promoter to the medium results in suppression of the expression of the recombinant polynucleotide encoding one or more biofilm assembly proteins and expression of the recombinant polynucleotide encoding a protease such that the bacterial host cell exhibits planktonic growth phase; and wherein the absence of the repressor for the first repressible promoter and the presence of the repressor for the second repressible promoter in the medium results in expression of the recombinant polynucleotide encoding one or more biofilm assembly proteins and suppression of the expression of the recombinant polynucleotide encoding a protease such that the bacterial host cell exhibits biofilm growth phase.
Narasaki teaches bacillolysin, a protease capable of breaking down the one or more biofilm assembly proteins “We isolated a novel protease … The protease, designated bacillolysin MA (BL-MA), belongs to a family of neutral metalloproteinases” (Abstract).
Narasaki does not teach (i) … a first repressible promoter; and
(ii) … a second repressible promoter;
wherein addition of a repressor for the first repressible promoter to the medium results in suppression of the expression of the recombinant polynucleotide encoding one or more biofilm assembly proteins and expression of the recombinant polynucleotide encoding a protease such that the bacterial host cell exhibits planktonic growth phase; and wherein the absence of the repressor for the first repressible promoter and the presence of the repressor for the second repressible promoter in the medium results in expression of the recombinant polynucleotide encoding one or more biofilm assembly proteins and suppression of the expression of the recombinant polynucleotide encoding a protease such that the bacterial host cell exhibits biofilm growth phase.
Taton teaches downregulation of gene expression in bacteria using “NOT gate” genetic circuits “To downregulate gene expression in cyanobacteria, we constructed NOT gate genetic circuits using orthogonal promoters and their cognate repressors regulated translationally by synthetic riboswitches.” (Abstract). Taton also teaches the modularity of their system “Further modification of these circuits could be achieved by using different promoters and riboswitches to drive expression of the repressors to produce variations in the levels of ON-state and OFF-state expression” (page 2179 2nd column, 2nd paragraph.
Taton teachings above teach the “wherein” clause of the claim. The “NOT gate” teaching of Taton, (use of orthogonal repressible promoters), within the Fernandez-Pitner-Narasaki’s planktonic/biofilm transition-dual orthogonal gene circuit system designs the resulting gene circuit to operate as described in the “wherein” clause of the claim.
It would have been obvious to a person having ordinary skill in the art (PHOSITA) before the effective filing date of the claimed invention to have combined Fernandez’s planktonic/biofilm transition system with Pitner’s dual orthogonal gene circuit system because Pitner provided motivation to modify their PASS platform “PASS presents a new strategy for engineering microbial gene regulation to achieve the desirable performance characteristics exhibited by natural mechanisms while also being amenable to modular protein and gene circuit engineering.” A PHOSITA would have followed Pitner’s teachings and further exploited its modularity within Fernanez’s planktonic/biofilm transition system.
Furthermore, it would have been obvious to a person having ordinary skill in the art (PHOSITA) before the effective filing date of the claimed invention to have substituted the TEV protease of Fernandez-Pitner’s planktonic/biofilm transition-dual orthogonal gene circuit system with Narasaki’s bacillolysin protease because it is a simple substitution of one known element for another to obtain predictable results. Narasaki’s bacillolysin protease is a functional protease and would function the same within Pitner’s dual orthogonal gene circuit system as outside of the system. Therefore a PHOSITA would have predicted the substitution of bacillolysin for TEV.
It would have been obvious to a person having ordinary skill in the art (PHOSITA) before the effective filing date of the claimed invention to have combined Fernandez-Pitner-Narasaki’s planktonic/biofilm transition-dual orthogonal gene circuit system with Taton’s teachings of downregulation of gene expression in bacteria using “NOT gate” genetic circuits because Taton provided motivation to modify their modular platform citing “Further modification of these circuits could be achieved by using different promoters and riboswitches to drive expression of the repressors to produce variations in the levels of ON-state and OFF-state expression.” Taton’s teachings would motivate a PHOSITA using Fernandez-Pitner-Narasaki’s system to use a first and second repressible promoter with the more biofilm assembly protein and protease capable of breaking down the one or more biofilm assembly proteins of Fernandez-Pitner-Narasaki. Fernandez, Pitner and Taton each demonstrated their gene circuit systems functioned, and further demonstrated their gene circuit systems were modular, and further taught their modular systems should be modified (as described above). Therefore, a PHOSITA would have a reasonable expectation of a functional modular gene circuit system with the aforementioned components of Fernandez-Pitner-Narasaki and Taton as described above.
Claim(s) 2 is/are rejected under 35 U.S.C. 103 as being unpatentable over Fernandez (Fernandez et al. Proc Natl Acad Sci U S A. 2020 Nov 17;117(46):29046-29054.), Pitner (Pitner et al. ACS Synth Biol. 2015 Sep 18;4(9):966-74.), Narasaki (Narasaki et al. J Biol Chem. 2005 Apr 8;280(14):14278-87.), and Taton (Taton et al. ACS Synth Biol. 2017 Dec 15;6(12):2175-2182.) as applied to claim 1 above, and further in view of Brodel (Brödel et al. Nat Commun. 2016 Dec 16;7:13858.).
Fernandez, Pitner, Narasaki, and Taton (Fernandez et al) teaches all of the elements of claim 1.
Fernandez et al do not teach the limitation “wherein the bacterial host cell additionally comprises: a recombinant polynucleotide encoding a protein operably linked to an inducible promoter for orthogonal expression in both biofilm growth phase and planktonic growth phase, wherein when an inducer is added to the medium, the bacterial host cell expresses the protein in both biofilm growth phase and planktonic growth phase.”
Brodel is in the same field of endeavor “Synthetic biology has seen an explosive growth in the capability of engineering artificial gene circuits from transcription factors (TFs), particularly in bacteria.” (Abstract). Brodel teaches “Design of a 3-input network. This network consists of three sensors, an integrated circuit with three cI variants operating on two unidirectional promoters, and two reporter genes.” (page 6, Figure 5c and legend; see image below). This passage and image teaches the orthogonal inducible promoter operably linked to a protein (Ara is the third inducer). It further teaches the expression in both biofilm and planktonic phase. Brodel teaches expression in biofilm and planktonic phase via orthogonal promoters; inducing the combination of 3OC6-Ara or IPTG-Ara. Furthermore, similar to Pitner and Taton, Brodel teaches the modularity of their genetic circuit system “Our toolkit contains 12 TFs, flexibly operating as activators, repressors, dual activator–repressors or dual repressor–repressors, on up to 270 synthetic promoters” (Abstract).
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It would have been obvious for a PHOSITA at the time of filing to have taken the combined system of Fernandez et al and added the third orthogonal gene circuit of Brodel because it is simply combining prior art elements according to known methods to yield predictable results. The instant limitation is simply an “OR gate” added to the gene circuit as described above and a PHOSITA would recognize it as such. Brodel teaches “protein operably linked to an inducible promoter for orthogonal expression.” Within Fernandez et al’s system, the expression could occur in both biofilm and planktonic phase as shown in the image (IPTG and 3OC6 would be the input for the other two elements of the orthogonal system). Similar to Fernandez et al’s genetic circuit described above, the modularity of Brodel’s circuit is such that each element individually functions the same when combined into the circuit. Therefore, a PHOSITA would have recognized the additional polynucleotide in the genetic circuit (of Brodel) would have predictable results.
Claim(s) 3 and 4 is/are rejected under 35 U.S.C. 103 as being unpatentable over Fernandez (Fernandez et al. Proc Natl Acad Sci U S A. 2020 Nov 17;117(46):29046-29054.), Pitner (Pitner et al. ACS Synth Biol. 2015 Sep 18;4(9):966-74.), Narasaki (Narasaki et al. J Biol Chem. 2005 Apr 8;280(14):14278-87.), and Taton (Taton et al. ACS Synth Biol. 2017 Dec 15;6(12):2175-2182.) as applied to claim 1 above, and further in view of Bermudes (Bermudes US 12537071 B1, effectively filed 2020 July 22).
Regarding claims 3 and 4, Fernandez, Pitner, Narasaki, and Taton (Fernandez et al) teach all of the elements of claim 1.
Fernandez et al does not the limitation “wherein the bacterial host cell additionally comprises a recombinant polynucleotide encoding a protein operably linked to the second repressible promoter for protein expression in planktonic growth phase.”
Bermudes is in the same field of endeavor of gene circuit design for engineered bacteria. For example, Bermudes teaches “Tumor-selective expression of therapeutic molecules by bacteria is achieved by one or more AND, NOR, OR, NOT and/or NAND gate genetic circuits. The therapeutic molecules can be proteins…” [Abstract].
Bermudes teaches the limitations of claims 3 and 4 by showing an “additional… polynucleotide encoding a protein” [in the image below, cldtB is 1st protein of claim 1, pltB is “additional… encoded protein” of claim 3, and pltA is “additional… encoded protein” of claim 4] under control of a single [second] promoter (in the image below, the lux promoter). Bermudes teaches, “The single or multiple open reading frames generated below may then be expressed … as components of a regulatory circuit as described in previous examples. Diagrams of targeted toxins are shown in FIGS. … 12A”[column 110, lines 40-44]; cytolethal distending toxin, CLDT; pertussis like toxin subunits, pltA and pltB.
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It would have been obvious for a PHOSITA at the time of filing to have taken the combined system of Fernandez et al and modified the second orthogonal gene circuit with Bermudes’s circuit because it is simply combining prior art elements according to known methods to yield predictable results. The instant limitation is simply an additional nucleotide sequence added to the reading frame of the “second promoter” of Fernandez et al’s gene circuit and a PHOSITA would recognize it as such. Similar to Fernandez et al’s genetic circuit described above, the modularity of Bermudes’s circuit is such that each element, individually, functions the same as when combined into the circuit. Therefore, a PHOSITA would have recognized the additional polynucleotide in the genetic circuit (of Bermudes) would have predictable results.
Claim(s) 5 is/are rejected under 35 U.S.C. 103 as being unpatentable over Fernandez (Fernandez et al. Proc Natl Acad Sci U S A. 2020 Nov 17;117(46):29046-29054.), Pitner (Pitner et al. ACS Synth Biol. 2015 Sep 18;4(9):966-74.), Narasaki (Narasaki et al. J Biol Chem. 2005 Apr 8;280(14):14278-87.), and Taton (Taton et al. ACS Synth Biol. 2017 Dec 15;6(12):2175-2182.) as applied to claim 1 above, and further in view of Kloosterman (Kloosterman et al. Mol Microbiol. 2007 Aug;65(4):1049-63.).
Regarding claim 5, Fernandez, Pitner, Narasaki, and Taton (Fernandez et al) teach all of the elements of claim 1.
Fernandez et al does not teach the limitation “wherein the second repressible promoter is PsczD and wherein the host cell additionally comprises a polynucleotide encoding a sczA operably linked to a PsczA promoter.”
Kloosterman is in the same field of endeavor of manipulating promoter-protein expression systems in bacteria. Kloosterman provides a figure of their engineered operon (gene circuit). The gene circuit comprises each element of the claim limitation, “the second repressible promoter is PsczD [also called PczcD, see [0092]],” and “the host cell additionally comprises a polynucleotide encoding a sczA operably linked to a PsczA promoter.” Kloosterman describes the engineered operon/gene circuit as follows “To be able to study the expression of the czcD operon in detail, an ectopic transcriptional lacZ fusion was constructed to the predicted promoter of czcD” (page 1050, 2nd column, 2nd paragraph, see Fig. 1A image below).
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Kloosterman’s Fig. 1 discloses the instant limitation, PsczD promoter sequence substituted for the “second promoter” of Fernandez et al’s gene circuit and “additional… polynucleotide encoding” the “sczA operably linked to a PsczA promoter.”
It would have been obvious for a PHOSITA at the time of filing to have taken the combined system of Fernandez et al and modified the second orthogonal gene circuit with Kloosterman’s circuit because it is simply combining prior art elements according to known methods to yield predictable results. Similar to Fernandez et al’s genetic circuit described above, the modularity of Kloosterman’s circuit is such that each element individually functions the same as when combined into the circuit. Therefore, a PHOSITA would have recognized Kloosterman’s PsczD promoter sequence operates as a promoter and would substitute for the “second promoter” of Fernandez et al’s gene circuit and therefore have predictable results. Similarly, Kloosterman’s “additional… polynucleotide encoding” the “sczA operably linked to a PsczA promoter operates as a promoter-protein system and therefore also have predictable results.
Claim(s) 6 is/are rejected under 35 U.S.C. 103 as being unpatentable over Fernandez (Fernandez et al. Proc Natl Acad Sci U S A. 2020 Nov 17;117(46):29046-29054.), Pitner (Pitner et al. ACS Synth Biol. 2015 Sep 18;4(9):966-74.), Narasaki (Narasaki et al. J Biol Chem. 2005 Apr 8;280(14):14278-87.), and Taton (Taton et al. ACS Synth Biol. 2017 Dec 15;6(12):2175-2182.) as applied to claim 1 above, and further in view of Llull (Llull et al. J Bacteriol. 2011 Apr;193(8):1919-29.).
Regarding claim 5, Fernandez, Pitner, Narasaki, and Taton (Fernandez et al) teach all of the elements of claim 1.
Fernandez et al does not teach the limitation “wherein the first repressible promoter is PzitR and wherein the bacterial host cell additionally comprises a polynucleotide encoding zitR operably linked to the PzitR promoter.”
Llull is in the same field of endeavor of manipulating promoter-protein expression systems in bacteria. Llull provides a figure of their operon (gene circuit). The gene circuit comprises each element of the claim limitation, “the first repressible promoter is PzitR [also called Pzn, see [0095]],” and “the host cell additionally comprises a polynucleotide encoding a zitR operably linked to a PzitR promoter.” Llull describes the operon/gene circuit as follows “L. lactis zitRSQP genes … are represented as large gray arrows. A putative ZitR repressor, ZitS lipoprotein, ZitQ ATP-binding cassette protein, and ZitP permease” (page 1920, 2nd column, 2nd paragraph, see Fig. 1A image below). Llull explains PzitR is equivalent to zitp “The zit promoter (zitp, previously referred to as PZn” (page 1920, Fig. 1 legend, Fig. 1A shown below).
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Llull’s Fig. 1 shows the instant limitation, zitp promoter (PzitR) sequence substituted for the “first promoter” of Fernandez et al’s gene circuit and “additional… polynucleotide encoding” the “ZitR operably linked to a PzitR promoter.”
It would have been obvious for a PHOSITA at the time of filing to have taken the combined system of Fernandez et al and modified the second orthogonal gene circuit with Llull’s circuit because it is simply combining prior art elements according to known methods to yield predictable results. Similar to Fernandez et al’s genetic circuit described above, the modularity of Llull’s circuit is such that each element individually functions the same as when combined into the circuit. Therefore, a PHOSITA would have recognized Llull’s zitp promoter sequence operates as a promoter and would substitute for the “first promoter” of Fernandez et al’s gene circuit and therefore have predictable results. Similarly, Llull’s “additional… polynucleotide encoding” the “ZitR operably linked to a PzitR promoter operates as a promoter-protein system and therefore also have predictable results.
Conclusion
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/AARON DUREL WARD/ Examiner, Art Unit 1636
/NEIL P HAMMELL/ Supervisory Patent Examiner, Art Unit 1636
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