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
The amendment of 06/13/2025 has been entered. Claims 1-6, 8-11, 14-35 are pending (claim set as filed on 06/13/2025). Claims 16-35 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claims 1-6, 8-11, 14-15 are currently under examination and were examined on their merits.
Information Disclosure Statement
The Information Disclosure Statement (IDS) filed on 06/13/2025 has been received and considered.
Withdrawn Objections/Rejections
The rejection of claim 4 under 35 U.S.C. 112(b) as being indefinite
as set forth in the previous Office action is withdrawn in light of the amendment filed on
06/13/2025.
All prior art rejections of claims 1-15 under 35 U.S.C. 103 set forth in the previous Office action are withdrawn in light of the amendment filed on 06/13/2025, which altered the scope of base claim 1. New rejections have been presented as discussed below.
Claim Rejections - 35 USC § 103
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.
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 factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
Determining the scope and contents of the prior art.
Ascertaining the differences between the prior art and the claims at issue.
Resolving the level of ordinary skill in the pertinent art.
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.
Claims 1-6, and 10 are newly rejected as necessitated by amendment under 35 U.S.C. 103 as being unpatentable over Bentley et al. (WO 2018/106932 A2; published on 06/14/2018), in view of Lin et al. (“Microfluidic technologies for studying synthetic circuits”; published on 05/12/2012, Current Opinion in Chemical Biology, Vol. 16, pages 307-317), Osmekhina et al. (“Controlled communication between physically separated bacterial populations in a microfluidic device, published on 07/20/2018, Communications Biology (2018), 1:97, pages 1-7; supplementary material, pages 1-9), and Woo et al. (“A designed whole-cell biosensor for live diagnosis of gut inflammation
through nitrate sensing”, published on 08/20/2020, Biosensors and Bioelectronics, Vol. 168, 112523, pages 1-10), as evidenced by Verbeke et al. (“Peptides as Quorum Sensing Molecules: Measurement Techniques and Obtained levels In vitro and In vivo”, published on 04/12/2017, Front. Neurosci., Vol. 11, Article 183, pages 1-18), and Kurkjian et al. (“Perspectives on the History of Glass Composition”, published 04/1998, J. Am. Ceram. Soc., Vol. 81, Issue 4, pages 795–813).
Bentley et al.’s general disclosure relates to “electrogenetic methods, devices and systems that use redox biomolecules to carry electronic information to engineered bacterial cells in order to control transcription from a synthetic gene circuit” (see entire document, including abstract).
Regarding claim 1, pertaining to the method of producing a recombinant protein, Bentley et al. teaches a method for producing a recombinant protein in a plurality of bacterial cells (“Electronic control of cell-to-cell communication … The receiver cells produce LuxR. When LuxR detects AHL, phiLOV is induced from the luxl promoter.“, “phiLOV fluorescent protein”, “The AHL receiver cell interprets the AHL cue by binding the LuxR protein and expressing phiLOV from the Vluxl promoter in the plasmid pTT06.”, “In electronically induced co-cultures, the AHL receiver cells exhibited an increase in fluorescence and emerged as a distinct fluorescent population”, “The majority of the experiments used E.coli DJ901 (…)”, “In situ electronic cell induction … An electrochemical setup as described above was used - with two chambers, three electrodes, and agar salt bridges. Cells at OD600 0.2 (…) were added to the working electrode vial”; “Electrochemical setup … the working and reference electrodes were placed in one glass vial with 3 mL of solution and/or cells”, “Induction of Cell-to-cell Communication…The cells were re-suspended in the M9 media at an OD600 of 0.25 and mixed at a 1:1 relay to receiver cell ratio before induction …Electrochemical induction was done as above with application of + 0.5 V for various times.”; page 4, line 21; page 5, lines 22-26; page 27, lines 2-4; page 27, lines 8-9; page 28, line 12; page 29, lines 19-30; page 30, lines 12-16; page 32, lines 1-8; see Fig. 5a; The Examiner notes that AHL is another name for autoinducer-1, as evidenced by Verbeke et al. (“Gram-negative bacteria use predominantly N-acyl homoserine lacton (AHL) molecules (autoinducer-1, AI-1)”; see entire document, including abstract), and that the substrate ‘glass’ comprises silica (SiO2) as evidenced by Kurkjian et al.(see entire document, including abstract and Table I on page 798)), said method comprising:
(a) contacting a first population of bacterial cells with a solid substrate in a culture vessel, said substrate comprising at least one exterior surface, at least one interior surface and at least on interior chamber defined by the interior surface and at least one opening (“the working and reference electrodes were placed in one glass vial with 3 mL of solution and/or cells”, “The cells were re-suspended in the M9 media at an 0D600 of 0.25 and mixed at a 1:1 relay to receiver cell ratio before induction.”; page 29, lines 29-30; page 32, lines 4-6; see vial in Figures 14a and 14b). The Examiner notes that the ‘glass vial’ taught by Bentley et al. corresponds to the substrate in a culture vessel recited in instant claim 1, since it has ‘at least one exterior surface, at least one interior surface and at least on interior chamber defined by the interior surface and at least one opening’ as recited in instant claim 1. The Examiner further notes that the ‘glass vial with 3 mL of solution and/or cells’ taught by Bentley et al. indicates that the cells are intrinsically contacted with the glass vial;
Bentley et al. further teaches
(b) applying a cell medium into the culture vessel (“The cells were re-suspended in the M9 media at an 0D600 of 0.25 and mixed at a 1:1 relay to receiver cell ratio before induction.”, “the working and reference electrodes were placed in one glass vial with 3 mL of solution and/or cells”; page 29, lines 29-30; page 32, lines 4-6; see Figures 14a and 14b),
(c) exposing the first population of bacterial cells with an inducer for a time period sufficient to stimulate expression of the recombinant protein (“Figure 5: Electronic control of cell-to-cell communication, (a) Schematic of electronic control of cell-to-cell communication. Electronic signals modulating Pyo and Fen (R) to Fen (0) result in Luxl-laa and AHL production from relay cells …The receiver cells produce LuxR. When LuxR detects AHL, phiLOV is induced from the luxl promoter. (b) Average fluorescence of biosensor cells within co-cultures in which relay cells are electronically induced with the indicated charges. (c) Flow cytometry histograms showing the emergence of a fluorescent receiver-cell population at the indicated time points after induction.”; page 5, lines 23-29; see Figure 5a-c). The Examiner notes that the increasing fluorescence of the receiver cells taught by Bentley et al. indicates that said cells were exposed with AHL for a time period sufficient to stimulate expression of the recombinant phiLOV fluorescent protein. Bentley et al. teaches wherein the inducer is produced from a second population of bacterial cells (“AHL production from relay cells”; page 5, line 24; see Fig. 5a).
Bentley et al. further teaches wherein the first population of bacterial cells comprises a nucleic acid molecule comprising an expressible nucleic acid sequence encoding the protein operably linked to a regulatory sequence specific for association with the inducer (“The AHL receiver cell interprets the AHL cue by binding the LuxR protein and expressing phiLOV from the Vluxl promoter in the plasmid pTT06; page 27, lines 2-4; see Figure 5a).
Additionally, Bentley teaches wherein AHL production in the second population of bacterial cells is induced by an external signal (“Electronic signals modulating Pyo and Fen (R) to Fen (0) result in Luxl-laa and AHL production from relay cells.”; page 5, lines 23-24).
Regarding claim 2, pertaining to transforming the first population of bacterial cells, Bentley et al. teaches wherein step (a) is preceded by transforming the first population of bacterial cells with the nucleic acid sequence (“The AHL receiver cell interprets the AHL cue by binding the LuxR protein and expressing phiLOV from the Vluxl promoter in the plasmid pTT06”; “Induction of Cell-to-cell Communication …The bioelectronic relay cells (DJ901 with the plasmid pTT05) and the receiver cells (DJ901 with the plasmid pTT06) were inoculated from overnight cultures”; “General Cloning Procedures … Electro- or chemically- competent cells (…) were used for transformation.”; page 27, lines 2-4; page 32, lines 1-3; page 35, lines 12-14; see Figure 5a).
Regarding claim 3, pertaining to the solid substrate, Bentley et al. teaches wherein the solid substrate comprises a base with a predetermined shape that defines the shape of the exterior and interior surface (see vial in Figures 14a and 14b).
Regarding claim 4, pertaining to the solid substrate, Bentley et al. teaches wherein the solid substrate comprises silica (“the working and reference electrodes were placed in one glass vial with 3 mL of solution and/or cells”; page 29, lines 29-30), and wherein the base is in shape of a cylinder prism, and wherein the silica coats the interior surface of the base and defines a cylindrical interior chamber (see vial in Figure 14b); Bentley et al. further teaches wherein the opening is positioned at one end of the cylinder (see the opening on one end of the cylinder prism (vial) in Figures 14a and 14b, where electrodes are inserted).
Regarding claim 5, pertaining to the inducer, Bentley et al. teaches wherein the inducer is autoinducer 1 (“When LuxR detects AHL, phiLOV is induced from the luxl promoter.“; page 5, lines 25-26; see Figure 5a). The Examiner notes that AHL is another name for autoinducer-1, as evidenced by Verbeke et al. (“Both Gram-negative and Gram-positive bacteria use this type of communication, though the signal molecules (auto-inducers) used by them differ between both groups: Gram-negative bacteria use predominantly N-acyl homoserine lacton (AHL) molecules (autoinducer-1, AI-1)”; see entire document, including abstract).
Regarding claim 6, pertaining to the nucleic acid molecule, Bentley et al. teaches wherein the nucleic acid molecule is free of secA, an extracellular secretion tag, and /or an outer membrane protein (“The AHL receiver cell interprets the AHL cue by binding the LuxR protein and expressing phiLOV from the Vluxl promoter in the plasmid pTT06”; “In embodiments, the encoded protein is a detectable protein. The detectable protein can be secreted, or non-secreted.”; page 13, lines 3-4; page 27, lines 2-4; see Table 1 on page 48, see pTT06 in Figure 8; note by Examiner, plasmid pTT06 does not indicate secA, an extracellular fusion tag, or an outer membrane protein).
Regarding claim 10, pertaining to an electrode, Bentley et al. teaches wherein an electrode is positioned within about 10 millimeters near the substrate (see Figure 14b). The Examiner notes that the vial taught by Bentley et al., which corresponds to the substrate of the instant application (as discussed above), has a diameter of about 1.5 cm (see Figure 14b), wherein the electrode is positioned inside the interior chamber close to the interior surface of the glass vial, therefore intrinsically positioned less than 10 mm from the interior surface. Bentley et al. further teaches a step of exposing the electrode to a voltage of 0.5 Volts (“Electrochemical induction was done as above with application of + 0.5 V for various times.”; page 32, lines 7-8).
Bentley et al. does not expressly teach wherein the volume of cell medium is sufficient to cover the at least one interior chamber (instant claim 1(b)).
Bentley et al. does not teach wherein the solid substrate further comprises a first, second, and third vessel; wherein each of the first, second and third vessels are of a size and shape sufficient to allow diffusion of protein, nutrients, and oxygen through the solid substrate in the presence of the cell culture medium; wherein the first and second vessels comprise the first and second populations of bacterial cells, respectively, and the third vessel comprises a third population of bacterial cells; wherein the third population of bacterial cells is capable of producing a signaling molecule that controls the production of the inducer in the second population of cells and wherein the first, second, and third vessels are in fluid communication (instant claim 1).
Lin et al.’s general disclosure relates to the “utility of microfluidics for the study of synthetic circuits” (see entire document, including abstract).
Regarding claim 1, pertaining to a substrate comprising three vessels and three cell populations, Lin et al. teaches a microfluidic device comprising a first, a second, and a third vessel, said vessels comprising a first, a second, and a third cell population, respectively (“Well established techniques exist for controlling cell positioning within microfluidic devices, including segregating cell populations into several discrete chambers”; page 308, left column, paragraph 1; see Figures 1 and 2f).
Osmekhina et al.’s general disclosure relates to a “microfluidic system that enables long-term and independent growth of fixed and distinctly separate microbial populations, while allowing communication through a thin nano-cellulose filter.” (see entire document, including abstract).
Regarding claim 1, pertaining to the solid substrate comprising multiple vessels, Osmekhina et al. teaches wherein a microfluidic device comprises a first and a second vessel, each vessel of a size and shape sufficient to allow diffusion of small molecules from one vessel to another in the presence of cell culture medium (“We constructed a polydimethylsiloxane (PDMS)-based microfluidic device, in which the growth of cells takes place in, and is confined to, a trapping chamber. This trapping chamber is divided into two parts by a filter made of rows of PDMS pillars and cellulose nanofibrils (CNFs)… entangled between them (…). The CNF layer functions as a filter separating two bacterial populations while allowing efficient signal exchange between them.”; “The CNF network remained porous enough to allow liquid and small molecules such as acyl-homoserine lactone (AHL) to pass through it.”; “cells were spun down, resuspended in 50 μL LB, and loaded into the microfluidic channels.”; page 2, left column, paragraph 6 - page 2, right column, paragraph 2; page 6, left column, paragraph 2; see Fig 2a and 2d).” ), and further teaches wherein the vessels are in fluid communication (“The CNF network remained porous enough to allow liquid and small molecules such as acyl-homoserine lactone (AHL) to pass through it.”; page 2, right column, paragraph 2).
Osmekhina et al. further teaches wherein the pore size between the vessels can be adjusted. (“depending on how the cellulose filter was prepared, the pore-size of the membranes could be adjusted.”; page 2, right column, paragraph 2). Osmekhina et al. further teaches wherein the method can be used to divide complex circuits over multiple populations which allows reusing regulatory elements and also reduces the expression burden of an individual cell population (“we have presented a method that enables the independent growth of two separated cellular populations …This allows multicellular strategies to divide complex circuits over multiple populations, dividing the genetic burden on each individual. Potential benefits of such an approach are in synthetic biology setups where genetic circuits are built to perform logic operations. … Logic gates can be constructed in bacteria using sets of genetic regulatory elements. To combine several gates within only one type of cell is generally technically challenging, partly because of the limited amount of regulatory elements that are available and suitable for building logic gates, and the overall expression burden on the bacterial host…. One problem is that the parts must be orthogonal, i.e., not interfering with other parts within a cell. Hence, wiring different cell types together is essential for building more complex systems, as the same part can be reused if separated into different cells. As such wiring between cell types can be done using signaling molecules such as AHLs, the dynamic communication shown in this study could lead to more efficient and responsive wiring, reduced individual burden, and hence better performing systems.” page 4, right column, paragraph 2 - page 5, left column, paragraph 1).
Additionally, Osmekhina teaches wherein an external signal such as arabinose or AHL can induce AHL production (“The sender cells produced a cyan fluorescent protein (CFP) and the Lux AHL in response to induction with arabinose”, “Three genes of the network, luxI, aiiA, and gfp, are under the control of the same lux promoter (pLux). LuxI produces AHL, which activates the transcription of all these genes in a complex with LuxR.; page 3, left column, paragraph 2; page 3 , right column, paragraph 2)
Woo et al.’s general disclosure relates to “a designer probiotic Escherichia coli that senses and responds to nitrate, a biomarker of gut inflammation” (see entire document, including abstract).
Regarding claim 1, pertaining to a third population of cells, Woo teaches “synthetic biology has provided various genetic circuits that allow microorganisms to sense input signals and respond to them via reprogramming cellular functions” (page 1 left column, paragraph 1), wherein the input signal can be an environmental signal (“Escherichia coli that senses and responds to nitrate”; see abstract).
While Bentley et al. does not expressly teach wherein the volume of cell medium is sufficient to cover the at least one interior chamber (instant claim 1(b)), the instantly recited volume would be within the realm of routine experimentation since Bentley et al. teaches wherein the interior chamber is filled with 3 mL of solution and/or cells and further comprises a working electrode that provides a voltage stimulus to the cells (see above). It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to determine the optimal amount of cell medium, in order to identify the optimal volume of cell medium for successful electrical induction of the cell system. Further, one would expect success since Bentley et al.’s teachings are directed to establishing a cell signaling system (see above), and therefore, manipulation of the volume of the cell medium based on the teachings of the reference would be within the purview of an artisan. Generally, differences in concentrations will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA1955). See MPEP § 2144.05 part II A.
While modified Bentley et al. does not teach wherein the solid substrate further comprises a first, second, and third vessel; wherein each of the first, second and third vessels are of a size and shape sufficient to allow diffusion of protein, nutrients, and oxygen through the solid substrate in the presence of the cell culture medium; wherein the first and second vessels comprise the first and second populations of bacterial cells, respectively, and the third vessel comprises a third population of bacterial cells; wherein the third population of bacterial cells is capable of producing a signaling molecule that controls the production of the inducer in the second population of cells and wherein the first, second, and third vessels are in fluid communication (instant claim 1), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined modified Bentley et al. with Bentley’s teachings on cells producing AHL in response to an external signal, Lin et al.’s teachings on a first, second and third vessel each comprising a first, second, and third population of cells (see above), Osmekhina et al.’s teachings on diffusion of small molecules from one vessel to another and on induction of autoinducer I production by autoinducer I (see above), and Woo’s teachings on bacteria responding to an external signal, in order to establish a method for producing a protein wherein the solid substrate further comprises a first, second, and third vessel; wherein each of the first, second and third vessels are of a size and shape sufficient to allow diffusion of protein, nutrients, and oxygen through the solid substrate in the presence of the cell culture medium; wherein the first and second vessels comprise the first and second populations of bacterial cells, respectively, and the third vessel comprises a third population of bacterial cells; wherein the third population of bacterial cells is capable of producing a signaling molecule that controls the production of the inducer in the second population of cells and wherein the first, second, and third vessels are in fluid communication. One would have been motivated to do so in order to establish a diffusion-based cell signaling system comprising a first, second, and third population of cells leading to efficient production of a recombinant protein in a first population of cells in response to an external signal sensed by a third population of cells. The resulting cell signaling system would allow for improved expression of a recombinant protein in a first population of cells due to an amplified inducer production generated by the second population in response to a signaling molecule produced by the third population of cells, while limiting the genetic and expression burden of an individual cell population (see Osmekhina et al. above). A skilled artisan would have expected success in the combination of Bentley et al.’s, Lin et al.’s, Osmekhina et al.’s and Woo et al.’s teachings, since all references are directed to engineered gene circuit systems (see above).
Claims 1 and 14 are newly rejected as necessitated by amendment under 35 U.S.C. 103 as being unpatentable over Bentley et al. (WO 2018/106932 A2; published on 06/14/2018), in view of Lin et al. (“Microfluidic technologies for studying synthetic circuits”; published on 05/12/2012, Current Opinion in Chemical Biology, Vol. 16, pages 307-317), Osmekhina et al. (“Controlled communication between physically separated bacterial populations in a microfluidic device, published on 07/20/2018, Communications Biology (2018), 1:97, pages 1-7; supplementary material, pages 1-9), and Woo et al. (“A designed whole-cell biosensor for live diagnosis of gut inflammation
through nitrate sensing”, published on 08/20/2020, Biosensors and Bioelectronics, Vol. 168, 112523, pages 1-10), as evidenced by Verbeke et al. (“Peptides as Quorum Sensing Molecules: Measurement Techniques and Obtained levels In vitro and In vivo”, published on 04/12/2017, Front. Neurosci., Vol. 11, Article 183, pages 1-18), and Kurkjian et al. (“Perspectives on the History of Glass Composition”, published 04/1998, J. Am. Ceram. Soc., Vol. 81, Issue 4, pages 795–813), in view of Lacoursiere et al. (“Effects of carbon dioxide concentration on anaerobic fermentations of Escherichia coli”, published 02/1986, Appl Microbiol Biotechnol (1986), Vol. 23, pages: 404-406).
Bentley et al.’s, Lin et al.’s, Osmekhina et al.’s and Woo et al.’s teachings have been set forth above.
Regarding claim 14, modified Bentley et al. teaches wherein a step of exposing the culture vessel to 37° Celsius and carbon dioxide leads to production of fluorescence in the first population of bacterial cells, therefore indicating that the culture vessel has been exposed for a time sufficient to allow expression of the protein in the first population of bacterial cells (“In situ Electronic Cell Induction … Cells were cultured as above and placed in the anaerobic chamber…Cells at OD600 0.2 (unless otherwise stated) were added to the working electrode vial and placed in the 37 °C mini incubator for ~ 5 min in order to warm before the addition of mediators. To initiate the electrochemical signaling, mediators were added, and the working electrode was biased at the indicated voltage for the indicated amount of time. For fluorescent cell sampling, about 100 μL of cells were removed from the solution”; “In electronically induced co-cultures, the AHL receiver cells exhibited an increase in fluorescence and emerged as a distinct fluorescent population”; “The receiver cells produce LuxR. When LuxR detects AHL, phiLOV is induced from the luxl promoter.“; “phiLOV fluorescent protein”; “In embodiments, cells that are subjected to electrical stimulation as described herein are maintained in anaerobic conditions, or hypoxic conditions, or aerobic conditions.”; “anaerobic chamber maintained anaerobic conditions - set up as per manufacturer's instructions, with nitrogen and CO2/H2/N2 mix”; page 4, line 21; page 5, lines 22-26; page 14, lines 29-31; page 27, lines 8-9; page 29, lines 2-4; page 30, lines 12-20; see Figure 5b). The Examiner notes that the increase of fluorescence in the receiver cells taught by Bentley et al. (see above) indicates that the culture vessel was exposed for a time sufficient to allow expression of the protein in the receiver cells. Modified Bentley et al. further teaches wherein the receiver cells is a strain of bacteria from Escherichia (“The majority of the experiments used E. coli DJ901”; “receiver cells (DJ901 with the plasmid pTT06)”; page 28, line 12; page 32, lines 2-3).
Modified Bentley et al. does not expressly teach wherein the culture vessel is exposed to a level of carbon dioxide of no more than about 5.0% (instant claim 14).
Lacoursiere et al.’s general disclosure relates to the effect of dissolved carbon dioxide concentration in the anaerobic growth of Escherichia coli (see entire document, including abstract).
Regarding claim 14, pertaining to the carbon dioxide, Lacoursiere et al. teaches wherein “E. coli was grown anaerobically under a variety of controlled dissolved CO2 concentrations” (page 404, right column, paragraph 2), and that there is “a sharp increase in both growth parameters as CO2 concentration increases from 0% to 0.025% in the gas phase. The maximum µ was 0.75 h -1 and occurred at 1.3 x 10-4 M CO2,
which corresponded to 5.0% carbon dioxide in output gas phase.” (page 406, left column, paragraph 1; see Fig. 3). LaCoursiere et al. further teaches that “enteric bacteria are probably especially adapted to the approximately 5% CO2 present in the gut of many mammals” (page 406, right column, paragraph 1).
While modified Bentley et al. does not teach wherein the culture vessel is exposed to a level of carbon dioxide of no more than about 5.0% (instant claim 14), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined the method taught by modified Bentley et al. with the teachings on optimal carbon dioxide concentrations for E.coli taught by Lacoursiere et al., in order to create a method for recombinant protein expression in E. coli wherein the culture vessel is exposed to a level of no more than 5% carbon dioxide. One would have been motivated to do so in order to provide optimal conditions for the E. coli cells for successfully expressing the recombinant protein. A skilled artisan would have expected success from the combination of modified Bentley et al.’s and Lacoursiere et al.’s teachings since both references are directed to E. coli under anaerobic conditions (see above).
Claims 1 and 15 are newly rejected as necessitated by amendment under 35 U.S.C. 103 as being unpatentable over Bentley et al. (WO 2018/106932 A2; published on 06/14/2018), in view of Lin et al. (“Microfluidic technologies for studying synthetic circuits”; published on 05/12/2012, Current Opinion in Chemical Biology, Vol. 16, pages 307-317), Osmekhina et al. (“Controlled communication between physically separated bacterial populations in a microfluidic device, published on 07/20/2018, Communications Biology (2018), 1:97, pages 1-7; supplementary material, pages 1-9), and Woo et al. (“A designed whole-cell biosensor for live diagnosis of gut inflammation
through nitrate sensing”, published on 08/20/2020, Biosensors and Bioelectronics, Vol. 168, 112523, pages 1-10), as evidenced by Verbeke et al. (“Peptides as Quorum Sensing Molecules: Measurement Techniques and Obtained levels In vitro and In vivo”, published on 04/12/2017, Front. Neurosci., Vol. 11, Article 183, pages 1-18), and Kurkjian et al. (“Perspectives on the History of Glass Composition”, published 04/1998, J. Am. Ceram. Soc., Vol. 81, Issue 4, pages 795–813), in view of Sonnenborn et al. (“The non-pathogenic Escherichia coli strain Nissle 1917 – features of a versatile probiotic”, published on 12/26/2009, Microbial Ecology in Health and Disease., Vol. 21, pages: 122–158).
Bentley et al.’s, Lin et al.’s, Osmekhina et al.’s and Woo et al.’s teachings have been set forth above.
Regarding claim 15, pertaining to a non-pathogenic strain of bacteria from Escherichia, modified Bentley et al. further teaches wherein the population of bacterial cells is a strain of bacteria from Escherichia (“The majority of the experiments used E.coli DJ901”; “receiver cells (DJ901 with the plasmid pTT06)”; page 28, line 12; page 32 lines 2-3). Additionally, Bentley et al. teaches “devices that can be biologic-based sensors that contain living cells that are maintained in a housing capable of sustaining the cells”(see abstract), wherein “the devices can be implantable or wearable” (see abstract), and further discloses that “a method, device and/or system of this disclosure … can thus be used in a wide variety of settings, including but not limited to medical devices, including but not necessarily limited to implantable medical devices,” (page 13, lines 15-18).
Modified Bentley et al does not expressly teach wherein the first population of bacterial cells is a non-pathogenic strain of bacteria from Escherichia (instant claim 15).
Sonnenborn et al.’s general disclosure relates to the non-pathogenic Escherichia coli stain Nissle 1917 (see entire document, including abstract).
Regarding claim 15, pertaining to a non-pathogenic strain of Escherichia, Sonnenborn et al. teaches a non-pathogenic strain of Escherichia (“E. coli strain Nissle 1917 (EcN)”, “With respect to its metabolic capacities, EcN is a typical E. coli strain. It is a non-pathogenic member of the Escherichia coli family, because it does not carry pathogenic adhesion factors and does not produce any enterotoxins or cytotoxins, it is not invasive, not uropathogenic, and is rapidly killed by non-specific defense factors of blood serum”; see abstract)
While modified Bentley et al. does not teach wherein the first population of bacterial cells are a non-pathogenic strain of bacteria from Escherichia (instant claim 15), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined the method of producing a recombinant protein taught by modified Bentley et al. with the non-pathogenic strain taught by Sonnenborn et al., in order to create a method wherein the first population of bacterial cells is a non-pathogenic strain of bacteria from Escherichia. One would have been motivated to do so, because modified Bentley et al. teaches using the recombinant protein production method in implantable medical devices (see above), therefore requiring non-pathogenic cells in order to protect a patient from potentially disease causing bacterial cells. A skilled artisan would have expected success in the combination of modified Bentley’s and Sonnenborn et al.’s teachings, since both references are directed to E. coli (see above).
Claim 1, 8, and 11, are newly rejected as necessitated by amendment under 35 U.S.C. 103 as being unpatentable over Bentley et al. (WO 2018/106932 A2; published on 06/14/2018), in view of Lin et al. (“Microfluidic technologies for studying synthetic circuits”; published on 05/12/2012, Current Opinion in Chemical Biology, Vol. 16, pages 307-317), Osmekhina et al. (“Controlled communication between physically separated bacterial populations in a microfluidic device, published on 07/20/2018, Communications Biology (2018), 1:97, pages 1-7; supplementary material, pages 1-9), and Woo et al. (“A designed whole-cell biosensor for live diagnosis of gut inflammation
through nitrate sensing”, published on 08/20/2020, Biosensors and Bioelectronics, Vol. 168, 112523, pages 1-10), as evidenced by Verbeke et al. (“Peptides as Quorum Sensing Molecules: Measurement Techniques and Obtained levels In vitro and In vivo”, published on 04/12/2017, Front. Neurosci., Vol. 11, Article 183, pages 1-18), and Kurkjian et al. (“Perspectives on the History of Glass Composition”, published 04/1998, J. Am. Ceram. Soc., Vol. 81, Issue 4, pages 795–813), in view of Rubens et al. (“Synthetic mixed-signal computation in living cells”, published on 06/03/2016, Nature Communications, 7:11658, pages 1-10, supplementary material pages 1-53),
Bentley et al.’s, Lin et al.’s, Osmekhina et al.’s and Woo et al.’s teachings have been set forth above.
Additionally, modified Bentley et al. teaches a nucleic acid encoding LuxI that allows cells comprising said nucleic acid to produce autoinducer 1 (AHL) in response to a redox signal controlled by an applied potential, wherein said redox signal comprises reduction-oxidation mediators (“Device-mediated electronic input consists of applied potential (…) for controlling the oxidation state of redox- mediators (...). Redox mediators intersect with cells to actuate transcription. … Fen (R/O), ferro/ferricyanide; Pyo, pyocyanin. The oxidation state of both redox mediators is colorimetrically indicated”; “Electronic signals modulating Pyo and Fen (R) to Fen (0) result in Luxl-laa and AHL production from relay cells”; “As seen in Figure 5a, in our relay cell, SoxR induces Vibrio fischerii Luxl (instead of phiLOV) expression from the plasmid pTT05. Luxl produces an acylated homoserine lactone (AHL), a bacterial signaling molecule that can diffuse through the membrane to guide quorum sensing (QS) behavior. The V. fischerii Luxl QS system has been widely used to engineer communication networks between non-communicating bacteria”; page 4, lines 8-17; page 5, lines 23-24; page 26, lines 30 – page 27, line 2; see Figures 5a and 8).
However, modified Bentley et al. does not teach wherein the first population of cells comprises a second expressible nucleic acid sequence encoding OxyR, wherein the nucleic acid sequence encoding OxyR is operably linked to a proD promoter sequence (instant claim 8), and further does not teach wherein the method is performed without exposure to reduction-oxidation mediators (instant claim 11).
Rubens et al.’s general disclosure relates to integrate analogue and digital computation to implement complex hybrid synthetic genetic programs in living cells (see entire document, including abstract).
Regarding claims 8 and 11, pertaining to a second expressible nucleic acid sequence (instant claim 8) and to a protein production method performed without exposure to reduction-oxidation mediators (instant claim 11), Rubens et al. teaches an expressible nucleic acid encoding OxyR, wherein the nucleic acid sequence OxyR is operably linked to a proD promotor sequence (instant claim 8) (see Supplementary Figure 1a). Rubens et al. further teaches an analogue sensor for hydrogen peroxide which comprises a constitutively expressed OxyR and teaches wherein OxyR oxidized by hydrogen peroxide induces expression of a gene regulated by the oxyS promoter wherein said induction does not require the exposure to reduction-oxidation mediators (instant claim 11) (“We first created an analogue sensor for the reactive oxygen species hydrogen peroxide (H2O2) … H2O2 oxidizes and activates the Escherichia coli transcription factor OxyR”; “OxyR is constitutively expressed from a low-copy plasmid (LCP) and activates transcription of gfp from the oxySp promoter on the same LCP in response to H2O2.”; page 2, left column, paragraph 3; see Supplementary Figure 1a). Rubens et al. further teaches wherein “H2O2 plays intricate biological roles across all kingdoms of life, and its regulation is linked to human health and disease.” (page 2, left column paragraph 3).
While modified Bentley et al. does not teach wherein the first population of cells comprises a second expressible nucleic acid sequence encoding OxyR, wherein the nucleic acid sequence encoding OxyR is operably linked to a proD promoter sequence (instant claim 8), and further does not teach wherein the method is performed without exposure to reduction-oxidation mediators (instant claim 11), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to have combined modified Bentley et al.’s method with Bentley et al.’s teachings on an expressable nucleic acid encoding LuxI and Ruben’s et al.’s teachings on the transcription regulator OxyR and a nucleic acid encoding OxyR, in order to create a first population of bacterial cells that comprises a second expressible nucleic acid sequence encoding OxyR, wherein the OxyR sequence is operably linked to a proD promoter, which leads to constitutive expression of OxyR (see Rubens et al. above), and wherein the first population of bacterial cells further comprises a third expressible nucleic acid encoding LuxI (see Bentley et al. above) wherein the expression of LuxI is induced by OxyR that has been oxidized by hydrogen peroxide (see Ruben’s et al. above). The expression of LuxI leads to AHL production as taught by Bentley et al. which induces production of the recombinant protein encoded by the first nucleic acid without exposure to reduction-oxidation mediators. One would have been motivated to do so in order to simplify the experimental setup and therefore improve the method for producing a recombinant protein by eliminating the reduction-oxidation mediators, but also to allow the first population of bacterial cells to produce AHL and therefore establish a communication system with other cells via AHL, since AHL diffuses through membranes (see Bentley et al. above). A skilled artisan would have expected success in the combination of modified Bentley et al.’s and Ruben et al.’s teachings since both references are directed to engineering gene circuits for E. coli (see above).
Claims 1 and 9 are newly rejected as necessitated by amendment under 35 U.S.C. 103 as being unpatentable over Bentley et al. (WO 2018/106932 A2; published on 06/14/2018), in view of Lin et al. (“Microfluidic technologies for studying synthetic circuits”; published on 05/12/2012, Current Opinion in Chemical Biology, Vol. 16, pages 307-317), Osmekhina et al. (“Controlled communication between physically separated bacterial populations in a microfluidic device, published on 07/20/2018, Communications Biology (2018), 1:97, pages 1-7; supplementary material, pages 1-9), and Woo et al. (“A designed whole-cell biosensor for live diagnosis of gut inflammation
through nitrate sensing”, published on 08/20/2020, Biosensors and Bioelectronics, Vol. 168, 112523, pages 1-10), as evidenced by Verbeke et al. (“Peptides as Quorum Sensing Molecules: Measurement Techniques and Obtained levels In vitro and In vivo”, published on 04/12/2017, Front. Neurosci., Vol. 11, Article 183, pages 1-18), and Kurkjian et al. (“Perspectives on the History of Glass Composition”, published 04/1998, J. Am. Ceram. Soc., Vol. 81, Issue 4, pages 795–813), in view of McKay et al. (“A platform of genetically engineered bacteria as vehicles for localized delivery of therapeutics: Toward applications for Crohn's disease”, published on 28 August 2018, Bioengineering & Translational Medicine 2018; Vol. 3, pages 209–221).
Bentley et al.’s, Lin et al.’s, Osmekhina et al.’s and Woo et al.’s teachings have been set forth above.
Additionally, Bentley et al. teaches “devices that can be biologic-based sensors that contain living cells that are maintained in a housing capable of sustaining the cells”(see abstract), wherein “the devices can be implantable or wearable” (see abstract), and further discloses that “a method, device and/or system of this disclosure … can thus be used in a wide variety of settings, including but not limited to medical devices, including but not necessarily limited to implantable medical devices,” (page 13, lines 17-18).
Additionally, Osmekhina et al. teaches an AHL-based positive feedback loop, wherein sender cells are induced by AHL to produce LuxI, which synthesizes AHL, and wherein AHL also induces expression of a recombinant protein in receiver cells (“To demonstrate the communication of bacteria physically separated by the CNF filter, we designed two populations of E. coli cells termed sender and receiver”; “We proceeded to demonstrate a dynamic communication through the CNF filter, where we studied the role of positive and negative feedback loops”; “In the sender population, expression of the luxI, aiiA, and gfp genes is controlled by the lux promoter. LuxI enzymatically produces AHL, which activates the lux promoter. The AHL lactonase, AiiA, hydrolyzes AHL, providing the negative feedback. AHL diffuses into the receiver cells and induce production of sfGFP and AiiA”; page 3, left column, paragraph 2; page 3, right column, paragraph 2; Figure 3a legend; see positive feedback loop in sender cells in Figure 3a; see abstract). Osmekhina et al. further teaches wherein the method can be used to divide complex circuits over multiple populations which allows reusing regulatory elements and also reduces the expression burden of an individual cell population (“we have presented a method that enables the independent growth of two separated cellular populations…This allows multicellular strategies to divide complex circuits over multiple populations, dividing the genetic burden on each individual. Potential benefits of such an approach are in synthetic biology setups where genetic circuits are built to perform logic operations. …Logic gates can be constructed in bacteria using sets of genetic regulatory elements. To combine several gates within only one type of cell is generally technically challenging, partly because of the limited amount of regulatory elements that are available and suitable for building logic gates, and the overall expression burden on the bacterial host…. One problem is that the parts must be orthogonal, i.e., not interfering with other parts within a cell. Hence, wiring different cell types together is essential for building more complex systems, as the same part can be reused if separated into different cells. As such wiring between cell types can be done using signaling molecules such as AHLs, the dynamic communication shown in this study could lead to more efficient and responsive wiring, reduced individual burden, and hence better performing systems.” page 4, right column, paragraph 2 - page 5, left column, paragraph 1).
However, modified Bentley et al. does not teach wherein upon exposure to an inducer, the first population of bacterial cells stimulates expression of a cytokine.
McKay et al.’s general disclosure relates to “engineered commensal E. coli that selectively synthesize and secrete a model biotherapeutic in the presence of nitric oxide” (see entire document, including abstract).
Regarding claim 9, pertaining to induced expression of a cytokine, McKay et al. teaches expression of GM-CSF in E. coli (“To assist in GM-CSF export, the pore-forming protein TolAIII is co-expressed in the engineered cells with a signal sequence that guides it to the outer membrane …Taken together, GM-CSF is first shuttled into the periplasm, and subsequently released into the extracellular space via the pores formed by TolAIII.”; “K-12 W3110 or Nissle 1917 E. coli (…) are used for all experiments for which data are presented.”; page 210, right column, paragraph 4; page 211, left column, paragraph 4; the Examiner notes that, according to the instant specification, the cytokine can be GM-CSF (“In some embodiments, the therapeutic molecule is a cytokine, such as GMCSF”; page 46, lines 28-29). Additionally, McKay teaches that “Previous reports indicate that recombinant GM-CSF produced in E. coli retains its biologic activity, despite lacking post-translational modifications such as N- and O-linked glycosylation.”; page 218, left column, paragraph 2), and further teaches that “The model biotherapeutic used in this study is granulocyte macrophage-colony stimulating factor (GM-CSF). This protein is chosen based on its reported therapeutic effects for individuals with CD,… and proven production in bacterial hosts… Briefly, CD is believed to have numerous contributing causes, comprising of a dysfunctional innate immune system (i.e., neutrophils) and a compromised mucosal barrier in the intestines.” (page 210, right column, paragraph 2; note, CD, Crohn’s disease, see abstract). McKay et al. further teaches that “this platform could be modified to accommodate other pursuits by swapping the promoter and therapeutic gene to reflect other disease biomarkers and treatments, respectively” (see abstract).
While modified Bentley et al. does not teach wherein, upon exposure to an inducer, the first population of bacterial cells stimulates expression of a cytokine (instant claim 9), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to have combined the modified method taught by Bentley et al. with the E. coli cytokine expression platform taught by McKay et al. and the AHL-based positive feedback loop taught by Osmekhina et al., in order to establish a first population of bacterial cells comprising an expressible nucleic acid encoding the recombinant protein luxI, wherein expression of said nucleic acid is induced by AHL as taught by Bentley et al. and Osmekhina et al. (see above). Expressed LuxI produces additional AHL, wherein the amplified AHL signal further induces expression of the cytokine in receiver cells comprising the E. coli cytokine expression platform taught by McKay et al., wherein the regulatory nucleic acid sequence associated with the gene encoding the cytokine is the lux promoter (see Osmekhin et al. above). One would have been motivated to do so in order to develop an improved treatment option for Crohn’s disease (see McKay et al. above), wherein the treatment device is an implantable medical device (see Bentley et al. above) comprising a cytokine producing receiver cell regulated by an AHL signal produced in sender cells. One would be motivated to use sender and receiver cells in order to reduce the genetic and expression burden on individual cell populations (see Osmekhina et al. above). A skilled artisan would have expected success in the combination of the above modified Bentley et al.’s, Osmekhina et al.’s and McKay et al.’s teachings since all references are directed to engineering genetic constructs using E. coli as a host.
Response to Arguments
Applicant has traversed all previous prior art rejections in light of the amendment of 06/13/2025, wherein claim 1 is amended to incorporate the limitations of previous claims