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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114 was filed in this application after a decision by the Patent Trial and Appeal Board, but before the filing of a Notice of Appeal to the Court of Appeals for the Federal Circuit or the commencement of a civil action. Since this application is eligible for continued examination under 37 CFR 1.114 and the fee set forth in 37 CFR 1.17(e) has been timely paid, the appeal has been withdrawn pursuant to 37 CFR 1.114 and prosecution in this application has been reopened pursuant to 37 CFR 1.114. Applicant’s submission filed on 12/31/2026 has been entered.
Appeal Board Decision
The Patent Board Decision mailed 11/03/2025 has AFFIRMED the Final Rejection mailed by the Office 4/30/2025. However, the Board recited a different rationale for the rejection of the claims, and hence issued new grounds of rejection of the claims. The Applicant has amended the present claims in response, and the present rejection is based on new grounds of rejection in light of the Applicant’s amendments. Where appropriate, the Board’s rationale has been incorporated into the present rejection of the claimed subject matter.
Application Status
This action is written in response to applicant’s correspondence received on 12/31/2026. Claims 1,4-20 and 22-27 are pending. Claims 1, 16, and 23 have been amended. Claims 2-3 and 21 have been cancelled. Claim 27 is newly added. All pending claims are currently under examination.
Claim Rejections - 35 USC § 103 – New Rejection Necessitated by Amendment
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.
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, 4-7, 10-14, 16-18, 20, and 23-27 are rejected under 35 U.S.C. 103 as being unpatentable over Ohlendorf (Ohlendorf R et al. J Mol Biol. 2012 Mar 2;416(4):534-42, Epub 2012 Jan 8. Erratum in: J Mol Biol. 2014 Jan 24;426(2):500, of record) in view of Marbach (Marbach A et al. J Biotechnol. 2012 Jan;157(1):82-8, of record), Falb (US 20170216370 A1, 2017, or record), Gardner (CA 2353800 A1, published 6/8/2000), and Collins (WO 2011/066541). The rejection of claim 5 is further evidenced by Lewis (Lewis M. The lac repressor. C R Biol. 2005 Jun;328(6):521-48, of record).
Regarding claim 1, Ohlendorf teaches the light-controllable system known as pDawn (Abstract). pDawn includes two sequences: a first sequence encoding a first repressor cI under the control of a first promoter FixK2, which is a light-controllable promoter (Figure 1). pDawn further includes a multiple-cloning site (MCS) for a practitioner to encode a second sequence (e.g., Ohlendorf encoded a second sequence, the antibiotic resistance gene CAT, into this site, Figure 5) which is under the control of a second promoter pR, which is controllable by the first repressor cI (Figure 1). Ohlendorf also teaches that the elements of pDawn are encoded in the pDawn plasmid (Abstract). Ohlendorf teaches and reduced to practice the use of the pDawn system in an engineered microorganism (Materials and Methods, Cell growth and fluorescence measurements section). Ohlendorf therefore teaches a repressor sequence present in a plasmid and introduced into an engineered microorganism.
Ohlendorf also teaches motivation to use their pDawn system to produce recombinant proteins and to replace conventional chemical induction methods using IPTG-inducible systems:
“as the performance of the light-inducible pDawn rivals that of the widely used pET system, pDawn can be used on a preparative scale for production of recombinant proteins. In comparison to induction by chemical means, for example, by IPTG, light induction is noninvasive, which reduces the risk of contamination, and cost-effective. Moreover, pDawn readily lends itself to automation: protein expression could be initiated at certain set points and adjusted by variation of time and intensity of illumination. In this manner, production yield and purity could be optimized, which is crucial in many areas including biotechnology and structural biology. Considering all these properties, we believe that pDawn will complement and, perhaps in certain cases, supersede conventional systems for induction of protein expression”, Discussion, first paragraph.
Ohlendorf therefore teaches that it would be superior to use the pDawn system, which is light-controllable, in a system that is normally induced by the chemical IPTG because the use of light-induction is more cost-effective, there is less risk of contamination, and protein expression can be optimized by varying illumination times (i.e., the system can be finely tuned based on time and intensity of light exposure, see quote above). Ohlendorf therefore reduced to practice use of their light-inducible system and teaches that it can “supersede” chemical induction methods (e.g., IPTG).
Ohlendorf therefore teaches a light-controllable single repressor system for the expression of a target gene (the pDawn system, above) and therefore does not teach a light-controllable two repressor system (the second sequence of Ohlendorf is not a repressor). Ohlendorf also does not teach a third sequence encoding the target gene under a third promoter, the third promoter controllable by the second repressor. Ohlendorf does not teach that the first promoter is controlled by the second repressor.
In addition, Falb, is in the field of engineered bacterial systems, teaches an inducible two-repressor system in genetically engineered bacteria, wherein the two-repressor system is a regulatory circuit designed to control the expression of a target gene: a propionate catabolism enzyme (paragraph 196). Falb teaches that their two-repressor system has at least three sequences: a first sequence encoding a first repressor (“comprises a first RNS-sensing repressor”, paragraph 196), a second sequence encoding a second repressor, (“a second repressor”, paragraph 196), and a third sequence encoding a target gene under a third promoter, the third promoter being controllable by the second repressor (“a second repressor, operatively linked to a gene or gene cassette, e.g., encoding a propionate catabolism enzyme”, paragraph 196). Falb therefore teaches two-repressor bacterial circuits with at least three sequences. Falb therefore teaches that two-repressor circuits are known in bacterially engineered systems.
Additionally, Falb teaches the use of the lac repressor and chemical induction using IPTG (paragraph 264), which are also taught by Ohlendorf (e.g., see page 539, left column, first paragraph for a discussion of IPTG and page 538, left column first paragraph for use of the lac operon/IPTG induction). Thus, Falb and Ohlendorf overlap in scope in the sense that they are bacterial engineered systems which teach the use of the same repressor and induction systems.
Furthermore, Falb teaches:
“the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a repressor-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a first repressor, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a payload operably linked to a first regulatory region comprising a constitutive promoter; and a third gene encoding a second repressor, wherein the second repressor is capable of binding to the first regulatory region and repressing expression of the second gene or gene cassette. The third gene is operably linked to a second regulatory region comprising a constitutive promoter, wherein the first repressor is capable of binding to the second regulatory region and inhibiting expression of the second repressor,” (paragraph 586).
Falb therefore teaches genetically engineered bacterial circuits comprising two repressor systems, comprising at least three sequences, including:
a first sequence encoding a first repressor, under control of a first promoter, the first promoter being an inducible promoter (“first gene encoding a first repressor, wherein the first gene is operably linked to a FNR-responsive promoter”)
a second sequence encoding a second repressor, under control of a second promoter, the second promoter being controllable by the first (“a third gene encoding a second repressor...The third gene is operably linked to a second regulatory region comprising a constitutive promoter, wherein the first repressor is capable of binding to the second regulatory region and inhibiting expression of the second repressor”)
; and a third sequence encoding the target gene under control of a third promoter, the third promoter being controllable by the second repressor (“a second gene or gene cassette for producing a payload operably linked to a first regulatory region comprising a constitutive promoter…a third gene encoding a second repressor, wherein the second repressor is capable of binding to the first regulatory region and repressing expression of the second gene or gene cassette” (Paragraph 586).
Falb therefore teaches inducible promoter gene circuits which differ from the claimed circuit only in that they are not light-inducible and that the second repressor does not repress the first promoter.
Falb teaches that the repressors can be lacI or cI, where cI is the same component used in the system of Ohlendorf (paragraph 587).
Falb further teaches Figure 24A, produced below:
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Falb teaches:
“FIG. 24A depicts another non-limiting embodiment of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the ParaBAD promoter (ParaBAD), which induces expression of the Tet repressor (TetR) and an anti-toxin. The anti-toxin builds up in the recombinant bacterial cell, while TetR prevents expression of a toxin ( which is under the control of a promoter having a TetR binding site). However, when arabinose is not present, both the anti-toxin and TetR are not expressed. Since TetR is not present to repress expression of the toxin, the toxin
is expressed and kills the cel,” (paragraph 32).
Falb therefore teaches that their circuits express heterologous target genes and are compatible with induction via exogenous environmental signals (paragraph 32).
In addition, Marbach, in the research field of IPTG induction of the lac operon in E. coli, teaches that IPTG is one of the most commonly used inducers in molecular biology and biotechnology (Introduction, second paragraph). Marbach also teaches that the expression of the lac operon is dependent on the inhibition of the repressor LacI, and that IPTG can be used to inhibit the action of the LacI repressor, thereby activating the lac operon (Introduction, first and second paragraphs). Marbach therefore teaches a second repressor (LacI) which binds to a third promoter/sequence (the lac operator).
Furthermore, Marbach teaches in the Abstract that “most commonly used expression systems in bacteria are based on the Escherichia coli lac promoter.” Marbach therefore teaches that the use of the lac operon is ubiquitous in the industry to express target genes, and therefore teaches high predictability when incorporating the lac operon into genetic circuits (third sequence under control of a third promoter where the third promoter is under control of the lacI repressor, the “second repressor”). As taught by Marbach, the use of the lac operon is ubiquitous in the industry and therefore teaches the industrial relevance of the lac operon (Abstract). Marbach therefore teaches promoter/repressor systems that would have been useful in a two repressor systems such as those taught by Falb, who also teaches the lac repressor and lac operon system (e.g., Falb paragraph 264, Marbach throughout and see pages 82-83).
Marbach teaches that “based on the detailed knowledge about the Escherichia coli lac operon and the easy controllability by chemical inducers, lac promoter
driven expression systems are favored in prokaryotes" (Marbach 82). Marbach discloses that "lac operon regulation is a research object in systems biology ... and lac operon elements are used as tools in biotechnology ...and synthetic biology" (Marbach 86; see also id. at Abstract). Marbach discloses that the "[m]ost commonly used expression systems in bacteria are based on the Escherichia coli lac promoter"… "[T]ranscription of the lac operon[, however,] is dependent on repression by the lactose repressor Lacl," wherein "[t]he most commonly used inducers [of the lac operon] in molecular biology and biotechnology are IPTG and TMG," which bind and inhibit Lacl repressor activity (Marbach 82).
Marbach teaches that induction of the lac operon by chemical means, such as IPTG, has drawbacks. For example, "it is known that IPTG can influence cell growth and induces a kind of stress response;" IPTG is modified by the action of transacetylase, which renders ... [it] unable to act as an inducer; and "the action of the transacetylase can lead to a reduction of the inducer concentration in the medium" (Marbach 87) and therefore teaches a known motivation to adopt other induction methods to induce the known lac operon, which relies upon the lacI repressor (above).
Regarding the limitation in claim 1 that the first promoter is “controllable by the second repressor” and that the second promoter is “controllable by the first repressor,” these genetic feedback loop designs of dual repressors controlling each other’s respective promoters are well-known in the art.
For instance, Gardner is a patent document which focuses on “on/off” expression of a gene of interest and therefore directly overlaps in subject matter and field of endeavor with Ohlendorf, Marbach, and Falb (see Abstract of Gardner). Furthermore, Gardner teaches that such designs as those with dual repressors under control of two different promoters, where each of repressor 1 and repressor 2 act to regulate the other’s promoter sequence are well known in the art as toggle-switches (e.g., Figures 1 and 20, Figure 1 reproduced below):
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Furthermore, Gardner supplies a direct motivation to incorporate such toggle switches with two repressors in control of each other’s promoters into genetic circuits because Gardner teaches that such dual repressors with such designs offer highly simplified and highly controlled models of gene networks (page 28, final paragraph). Thus, Gardner teaches that such dual repressor designs where each repressor controls the opposing promoter are useful in genetic circuit designs, offering superior/tighter control of the expression of a desired product (page 28, final paragraph, Figure 1).
In addition, Gardner teaches that such designs can be expanded to include higher order genetic circuits including three sequences, including designs where dual repressors control each other’s respective promoters, where furthermore an additional third construct/sequence with promoter controlling another gene of interest is controlled by the second repressor (see Figure 9C):
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Thus, Gardner teaches that such dual repressor systems can be modulated, and further teaches the same components as Falb to be used such as the lacI and CI components (page 11, first paragraph), where Gardner teaches useful genetic circuit designs comprising three constructs with dual repressors controlling opposing promoters and an additional third construct and gene (Figure 9, e.g., Figure 9C).
Additionally, Gardner teaches that the “on/off” switch mechanism can be controlled by light (page 30, first paragraph), and therefore directly teaches that the environmental signal input controlling the circuit can be an optogenetic circuit similar to those taught by Ohlendorf (page 30, first paragraph).
Furthermore, Collins is a patent document which focuses on genetic circuits and their uses in various applications (Title, Abstract, and throughout). Collins teaches toggle-switching genetic circuits which rely upon a first promoter expressing a first repressor which controls a second promoter, where the second promoter expresses a second repressor which controls the first promoter(e.g., paragraph 83). Collins therefore also teaches dual repressor genetic circuits systems where the first promoter expresses a repressor which regulates the expression of second promoter which expresses a second repressor, where the first and second repressors regulate each other’s promoter via a feedback loop (paragraph 83). Furthermore, Collins teaches that:
“several accessible technologies for increasing the throughput and pace of piece-wise gene-circuit characterization. Recent advances in engineering light-inducible biological parts and systems have unlocked the potential for optical-based circuit characterization. For instance, by coupling a synthetic gene network of interest to light-inducible systems as well as fluorescent protein outputs, both control and monitoring could be accomplished via the reliable and high-speed optics that are typically associated with fluorescence microscopy. This prospect, particularly in the context of microfluidic devices, can facilitate the focusing of optical inputs and read-outs to single cells,” (paragraph 288).
Thus, Collins further teaches that coupling gene circuits specifically with optically controlled circuits has added benefits such as reliability and improved control (above). Thus, the art is not only replete in knowledge when using dual repressor feedback systems such as those recited, but coupling of such genetic networks and circuits to be used with light-inducible optogenetic circuits such as those taught by Ohlendorf has been directly suggested by both Collins and Gardner, who teach that combining such circuits would render improved control and reliability (paragraph 288 of Collins, paragraph 1 of page 30, Gardner).
Collins teaches that their circuit designs are “highly modular and can function with a variety of combinations of various component parts, such as toggle switches and recombinases. Depending on the combinations of modules and component parts used in the biological chemotactic converters described herein, a chemotactic converter can respond to one or to multiple inputs,” (paragraph 8). Collins therefore teaches that their designs are inherently modular and compatible with combinations of various elements including toggle switches with dual repressors regulating each other’s promoters, where the input responses can be any number of inputs. Collins teaches that the input to control the circuits can be light (paragraph 81, 266).
Furthermore, Collins teaches a direct motivation to incorporate “feedback” systems within genetic circuits, such as the dual repressor feedback loop taught by Falb (Collins, paragraph 7). For instance, Collins teaches that:
“[a]chieving robustness, an important property of engineered systems, requires carefully incorporation of the concepts of feedback, redundancy, and decoupling. For example, robustness in a synthetic gene oscillator composed of linked negative and positive feedback loops was found to be dependent on a time delay in the negative feedback loop. To achieve robustness, synthetic biologists need to adapt designs from natural systems, incorporate feedback and multiple redundant circuits, and seek out evolutionary methods to refine circuit performance. Accelerated, large-scale diversification and the use of characterized component libraries in conjunction with in silica models for a priori design help to fine-tune network performance toward the desired output,” (paragraph 278).
Thus, Collins teaches that there is a motivation known in the art to incorporate feedback mechanisms into the design of genetic circuits, where such designs can be achieved via modeling, and furthermore such designs help to fine-tune the network performance for a desired output (paragraph 278).
Additionally, Collins teaches that such genetic circuits can be modified and expanded to generate multi-layered genetic circuits comprising multiple repressor systems (Figure 5B) and/or to regulate and express a desired protein (Figure 8, GFP). Thus, Collins teaches that such multiple repressor systems with feedback loops using two repressors with modular designs are within the design capacity of a person of ordinary skill in the art because gene circuits with even higher order complexity than those presently recited are already known in the art to be functional (Collins, Figure 5B, Figure 8).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the pDawn system disclosed by Ohlendorf with the teachings of Marbach, Falb, Gardner, and Collins to arrive at the present invention because such a combination is the simple combination of known prior art elements with predictable success. Specifically, considering Ohlendorf’s pDawn system in the context of Falb, a person of ordinary skill in this art would have found it prima facie obvious to utilize the YFl/FixJ TCS of pDawn to drive expression of the lambda phage repressor cl (i.e. a first repressor) that is under the control of the light-controllable pFixK2 promoter (i.e. first promoter), which, in tum, represses expression
from the strong lambda promoter pR (i.e. second promoter controllable by the first
repressor ), which would drive the expression of a second repressor that controls the expression of a target gene under the control of a third promoter (i.e. a target gene under control of a third promoter that is controllable by the second repressor) where such a two repressor system would achieve a desired gene expression goal, as taught by Falb. Furthermore, the limitations that the first promoter expresses a repressor that controls a second promoter, where the second promoter expresses a repressor that controls the first promoter, is obvious in view of the teachings of Gardner and Collins, where such repressor feedback loops are well-known in the art, where furthermore such feedback loops have been suggested to be used with light-inducible genetic circuits and offer advantages such as higher degrees of control for such genetic circuits and better ability to fine-tune the expression of a desired output product. Thus, the practitioner is motivated to incorporate the teachings of Gardner and Collins’ dual repressor systems with Ohlendorf and the dual repressor systems of Falb because Gardner and Collins teach direct motivation to use such controlled gene circuits for fine-tuned control of dual repressor circuits. The result of such a combination would be a circuit with light-inducible promoters such as those taught by Ohlendorf, where the dual repressor system of Falb would be modified so that the first promoter is controlled by the second repressor, as Gardner/Collins teaches that this is a useful way to control the expression of desired gene products, the goals of both Ohlendorf and Falb.
Additionally, integration into the genome is not required for the broadest reasonable interpretation of claim 1, which allows that, alternatively, “at least one sequence” is present in a plasmid. As stated above, Ohlendorf teaches at least one sequence integrated in a plasmid in an engineered microorganism. An obvious rationale for integrating at least one of the sequences into the genome of an engineered microorganism is therefore not required in order to reject claim 1 under 103.
Furthermore, promoter and repressor elements such as lacI, the lac operon, and cI are well-known, where the combination is therefore predictable (e.g., the teachings of Marbach, pages 82-83).
Thus, when considering the totality of what is known in the art surrounding genetic circuits, the present invention is rendered obvious by the combination Ohlendorf, Falb, Marbach, Gardner, and Collins, where each element is known, and furthermore suggestions and motivation to use three-construct, dual repressor systems such as those taught by Falb, in combination with feedback repressor designs offering tighter control, are taught by Gardner and Collins. The art is therefore replete with knowledge surrounding dual-repressor constructs used in genetic circuits, where even higher order circuits are known and have been reduced to practice and are therefore predictable.
Regarding claim 4, Ohlendorf teaches the first repressor phage repressor cI (Figure 1). Marbach teaches the second repressor LacI (Introduction, lines 15-16). For reasons discussed above, it would be obvious to combine the teachings of Marbach and Ohlendorf to arrive at claim 4.
Regarding claim 5, Marbach teaches the lac operon but does not teach that the lac promoter is a lacO-operator-containing promoter. As evidenced by Lewis (Background, paragraph 5), the lac operon includes a lacO-operator binding site. The lac operon taught by Marbach therefore inherently includes a lacO-operator-containing promoter.
Regarding claim 6, Ohlendorf teaches that the first sequence encoding the repressor cI further encodes a C-terminal LVA tag in the pDawn system, which decreases the intracellular lifetime of the repressor (Materials and Methods, “Construction of pDusk and pDawn,” second paragraph). Ohlendorf therefore teaches degradation tags of the first repressor.
Regarding claim 7, Ohlendorf teaches that the first promoter is pFixK2 (Figure 1).
Regarding claim 10, Ohlendorf teaches that “pDawn can be used on a preparative scale for production of recombinant proteins,” Discussion, first paragraph, lines 13-14.
Regrading claim 11, Ohlendorf teaches that DsRed, a red fluorescent protein, was used in their study as the target gene to be expressed by the pDawn system (Figure 2).
Regarding claim 12, Ohlendorf teaches that the elements of pDawn are encoded in the pDawn plasmid (Abstract).
Regarding claim 13, Ohlendorf uses the pDawn system in a microorganism (Materials and Methods, Cell growth and fluorescence measurements section).
Regarding claim 14, Ohlendorf uses the pDawn system in E. coli (Materials and Methods, Cell growth and fluorescence measurements section).
Regarding claim 16, the physical components recited in claim 16 are identical to those recited in claim 1 and are addressed above in the rejection of claim 1. Rationales and motives to create such a system are discussed above for claims 1 and 4-7, and10-14. Furthermore, bacterial expression systems using two repressors were known in the art, as evidenced by Falb, Gardner, and Collins as discussed above. Additionally, Ohlendorf teaches the method of growing cultures that are grown in a first lighting condition and then adjusting the lighting condition to allow the induction of a target gene (Materials and Methods, section “Cell growth and fluorescence measurements,” second paragraph). Furthermore, Figure 6 of Ohlendorf shows a method where the pDawn system is grown and expressed under conditions using blue-light pulses, wherein a blue light was turned on for a time T1 and then turned off for a time T2 during a 5-hour incubation period. Ohlendorf therefore teaches adjusting the first lighting condition by turning on a light source for a first time period T1 then turning off the light source for a second period of T2. Additionally, Ohlendorf teaches that their system should be optimized to express target recombinant proteins by teaching that:
“pDawn readily lends itself to automation: protein expression could be initiated at
certain set points and adjusted by variation of time and intensity of illumination. In this manner, production yield and purity could be optimized”, page 539, left column, first paragraph.
Regarding claim 17, Ohlendorf teaches growth conditions where cultures are first grown in a non-inducing lighting condition, wherein said lighting condition is adjusted when the OD600 value of the microorganism was at a predetermined value (Materials and Methods, section entitled “Cell growth and fluorescence measurements,” second paragraph).
Regarding claim 18, the OD600 value in the Ohlendorf method is 0.4, which falls within the range given in claim 18 (Materials and Methods, section entitled “Cell growth and fluorescence measurements,” second paragraph).
Regarding claim 20, Ohlendorf teaches that “pDawn can be used on a preparative scale for production of recombinant proteins” (Discussion, first paragraph). Ohlendorf also teaches that the protein of interest DsRed is purified by collecting and lysing cells in a culture and obtaining the purified protein of interest (section entitled “Protein Purification”).
Regarding claim 23, claim 23 is simply a logical embodiment of the claimed elements of the disclosed invention in order to use the claimed invention, all of which elements are obvious in view of Ohlendorf and Marbach, Falb, Gardner, and Collins (see rejection of claim 1). A practitioner could immediately envision supplying the components in a kit.
Regarding claim 24, as discussed above, a combination of Ohlendorf, Marbach, Falb, Gardner, and Collins yields the two-repressor light-controllable system of claim 1. Furthermore, Gardner teaches that the first nucleotide sequence can comprise a sequence downstream from the first promoter that is controllable by the second repressor (see Figure 17 of Gardner). Thus, the presently recited subject matter could be arrived at by the known design principles taught in the art according to the combination of Ohlendorf, Marbach, Falb, Gardner, and Collins.
Regarding claim 25, Ohlendorf teaches the use of degradation tags in their system, and states that, with regards to the cI repressor they used, their degradation tag on said repressor “greatly decreases its intracellular lifetime, to improve the response dynamics of the inverted system,” Materials and Methods, Construction of pDusk and pDawn, second paragraph. Ohlendorf therefore teaches that adding degradation tags to repressors can improve the response dynamics of genetically engineered systems.
Furthermore, Collins also teaches that degradation tags can be added to the proteins of their circuit designs (paragraph 209), where furthermore Collins also teaches that their circuit designs comprise multiple repressors (e.g., Figure 5B).
Therefore, it would be obvious to include a degradation tag on the second repressor because, as taught by Ohlendorf/Collins, by so doing a practitioner would improve the response dynamics of a light-controllable system.
Regarding claim 26, Ohlendorf teaches that:
“pDawn readily lends itself to automation: protein expression could be initiated at
certain set points and adjusted by variation of time and intensity of illumination. In this manner, production yield and purity could be optimized”, page 539, left column, first paragraph.
Thus, Ohlendorf teaches that protein expression levels can be adjusted or tuned based on the time and intensity of illumination, and further that such adjustments can be changed to optimize production yields (page 539, left column, first paragraph): Ohlendorf therefore teaches different T1 and T2 selection times for the expression of target genes because they teach that such times can be adjusted in order to optimize their methods.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to use the system taught by Ohlendorf to express target genes for a longer time period than non-expression of the target gene, because such claim limitations are simply the routine optimization of the method taught by Ohlendorf. As discussed above, Ohlendorf teaches that their method should be optimized to express proteins by adjusting the time and the intensity of illumination (page 539, left column, first paragraph). Therefore, a practitioner would be motivated to optimize target gene expression in the systems taught by Ohlendorf. The claim limitations recited in claim 26 would therefore be arrived at by routine optimization of Ohlendorf’s method and teachings.
Regarding claim 27, Ohlendorf teaches the use of degradation tags in their system, and states that, with regards to the cI repressor they used, their degradation tag on said repressor “greatly decreases its intracellular lifetime, to improve the response dynamics of the inverted system,” Materials and Methods, Construction of pDusk and pDawn, second paragraph. Ohlendorf therefore teaches that adding degradation tags to repressors can improve the response dynamics of genetically engineered systems.
Furthermore, Collins also teaches that degradation tags can be added to the proteins of their circuit designs (paragraph 209), where furthermore Collins also teaches that their circuit designs comprise multiple repressors (e.g., Figure 5B).
Given that Ohlendorf has already taught degradation tags which can be added to the repressors of their systems, and furthermore that Collins also teaches that degradation tags can be added to their systems which comprise multiple repressors, it would be obvious to a person of ordinary skill in the art to include such degradation tags on the second repressor, simply by design choice and optimization of the systems taught by Ohlendorf, where Ohlendorf teaches the strategy of adding degradation tags in order to have a desired, beneficial outcome in their systems (” Materials and Methods, Construction of pDusk and pDawn, second paragraph). For instance such tags could be used to more finely tune the response of the systems of Ohlendorf, as Ohlendorf has also taught (above). The claim limitations are further obvious given that Collins has also suggested and taught the use of degradation tags on the components of their genetic circuits, where their circuits comprise multiple repressors (e.g., Figure 5B). Hence, the addition of a degradation tag to the second repressor is obvious in view of the teachings of both Ohlendorf and Collins.
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Ohlendorf (Ohlendorf R, Vidavski RR, Eldar A, Moffat K, Möglich A. From dusk till dawn: one-plasmid systems for light-regulated gene expression. J Mol Biol. 2012 Mar 2;416(4):534-42. doi: 10.1016/j.jmb.2012.01.001. Epub 2012 Jan 8. Erratum in: J Mol Biol. 2014 Jan 24;426(2):500) in view of Marbach (Marbach A, Bettenbrock K. lac operon induction in Escherichia coli: Systematic comparison of IPTG and TMG induction and influence of the transacetylase LacA. J Biotechnol. 2012 Jan;157(1):82-8), Falb (US Patent US 20170216370 A1, 2017), Gardner (CA 2353800 A1, published 6/8/2000), and Collins (WO 2011/066541 as applied to claim 1 above, and further in view of Zucca (Zucca S, Pasotti L, Politi N, Cusella De Angelis MG, Magni P. A standard vector for the chromosomal integration and characterization of BioBrick™ parts in Escherichia coli. J Biol Eng. 2013 May 10;7(1):12).
As discussed above, the combination of Ohlendorf, Marbach, Falb, Gardner, and Collins teaches the system of claim 1, wherein sequences of the system are found in a plasmid.
Ohlendorf, Marbach, Falb, Gardner, and Collins do not teach or suggest that sequences of system 1 are integrated into the genome of the engineered microorganism.
Zucca, in the research field of recombinant E coli cells, teaches that “genome integration can provide the stable insertion of the desired genes in the host chromosome without the need of any antibiotic or resistance marker,” Background, first paragraph. Zucca therefore teaches that integrating components of genetic systems into an organism’s genome provides a method to stably introduce a desired gene into said system.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify light-inducible, lacI, two-repressor system taught by Ohlendorf, Marbach, Falb, Gardner, and Collins by integrating at least one of the sequences in this system into the genome of the engineered microorganism because Zucca teaches that doing so confers an advantage to the system, namely, that it provides a means to stably introduce a gene into a genetic system.
Claims 8,9, and 19 are rejected under 35 U.S.C 103 as being unpatentable over Ohlendorf, in view of Marbach and Falb, Gardner (CA 2353800 A1, published 6/8/2000), and Collins (WO 2011/066541) as applied to claims 1 and 16 above, and further in view of Martin 1(Martin VJ, Pitera DJ, Withers ST, Newman JD, Keasling JD. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol. 2003 Jul;21(7):796-802). Claim 19 is further evidenced by Martin 2 (Martin VJ, Yoshikuni Y, Keasling JD. The in vivo synthesis of plant sesquiterpenes by Escherichia coli. Biotechnol Bioeng. 2001 Dec 5;75(5):497-503).
Regarding claims 8, and 9, Ohlendorf, Marbach, Falb, Gardner, and Collins teach the elements of claim 1, as discussed above. Ohlendorf, Marbach, Falb, Gardner, and Collins do not teach that the target gene is involved in the biosynthesis of a chemical compound, wherein the chemical compound is mevalonate or isobutanol.
Martin 1, in the research field of metabolic engineering in E. coli, teaches an IPTG-inducible operon for the mevalonate isoprenoid pathway in E. coli (Figure 1, and the “Construction of the mevalonate pathway operons” section of the Methods section). Martin also teaches that isoprenoids are an important commercial product used in fragrances, flavors, and antimalarial and anticancer drugs (Abstract).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to further modify the teachings of Ohlendorf Marbach, Falb, Gardner, and Collins with the IPTG-inducible system taught by Martin 1. Ohlendorf stated that a light-inducible system would be advantageous over an IPTG-inducible system. The motivation for a practitioner to make the system taught by Martin 1 under control of the system taught by Ohlendorf is supplied by Ohlendorf, who, as discussed above for the rejection of claim 1, has stated that light-controllable systems offer advantages over chemical-inducible systems. The motive to adapt the mevalonate isoprenoid pathway expression system taught by Martin 1 to work with the light-controllable system taught by Ohlendorf specifically is that the mevalonate isoprenoid pathway system yields commercially relevant products as discussed in the Abstract of Martin 1.
Regarding claim 19, Martin 1 teaches that amorphadiene production was analyzed using a method taught by Martin 2 after cells were induced to express the mevalonate pathway (Martin 1, “GC-MS analysis of amorphadiene”). Martin 2 teaches the extraction of sesquiterprenes from culture aliquots. Martin 1, who incorporated the reference Martin 2, therefore taught obtaining a cell-free supernatant containing a chemical of interest expressed by a target gene because the sesquiterpenes were extracted from culture aliquots (Martin 2, “GC-MS Analysis of Sesquiterpenes”).
Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over Ohlendorf, Marbach, Falb, and Gardner (CA 2353800 A1, published 6/8/2000), and Collins (WO 2011/066541 as applied to claim 16 above, and further in view of Jayaraman (Jayaraman P, Devarajan K, Chua TK, Zhang H, Gunawan E, Poh CL. Blue light-mediated transcriptional activation and repression of gene expression in bacteria. Nucleic Acids Res. 2016 Aug 19;44(14):6994-7005. doi: 10.1093/nar/gkw548. Epub 2016 Jun 28).
A combination of the teachings of Ohlendorf, Marbach, Falb, Gardner, and Collins arrive at the method described in claim 16, as discussed above. However, Ohlendorf, Marbach, Falb, Gardner, and Collins do not teach that T1/(T1 +T2) is between about 0.001 and about 0.1.
Jayaraman, in the research field of blue-light induced bacterial expression systems, teaches a light-cycling method to induce the expression of a protein with blue light wherein T1 = 5 seconds, T2 = 55 seconds, and T1/(T1 +T2) = 5/(5 +55) = 5/60 = ~0.083, which is within the range recited in claim 22 (Figure 2 of Jayaraman).
It would have been obvious to one of ordinary skill in the art before the time of the effective filing date of the claimed invention to modify the teachings of Ohlendorf, Marbach, Falb, Gardner, and Collins with the light-cycling method taught by Jayaraman. Jayaraman teaches that pulses of light cycles can be used to finely tune gene expression in blue-light inducible bacterial expression systems in order to precisely control gene expression (Abstract). Furthermore, Jayaraman teaches that “a rapid increase in expression of the blue light inducible system is seen when the blue light pulse ON–OFF cycle increases from 0 to 8.33% (5 s ON; 55 s OFF)” (“Dose-dependent activation and repression,” first paragraph). Claim 22 is therefore merely a combination of elements taught by Ohlendorf, Marbach, Falb, Gardner, and Collins with a method taught by Jayaraman with predictable results.
Response to Arguments
The Applicant’s arguments filed 12/31/2025 have been considered but are not persuasive. The Applicant argues that their amendments to the claims place the claims in condition for allowance. This argument is not persuasive because the amendments prompted a new search which uncovered Gardner and Collins. As discussed in detail above, the amendments to the claims with respect to the limitation “the first promoter is controllable by the second repressor” is obvious in view of the combinations of Ohlendorf, Marbach, Falb, Gardner, and Collins. The art is replete with knowledge surrounding genetic circuits comprising multiple repressors (Falb, Gardner, Collins), where furthermore the kinds of feedback loops presently recited, where repressors 1 and 2 act upon each other’s respective promoter to regulate each other’s expression is a known approach to genetic circuit engineering per Gardner and Collins, where furthermore Gardner and Collins provide ample motivation to combine such useful genetic feedback circuits with dual repressor systems such as those taught by Falb and also light-inducible systems such as those taught by Ohlendorf (see art rejection above). As such, a person of ordinary skill in the art could arrive at such circuits by combining the known prior art elements with predicable success, where motivation exists to create tightly regulated gene expression, per Gardner and Collins. Additionally, Collins directly suggests using such dual repressor toggle switches in combination with optogenetic circuits; the combination would therefore function predictably, as it is known that such toggle switch genetic circuits as those taught by Gardner/Collins are compatible with optogenetic/light-inducible circuits as taught by Ohledndorf (above, rejection of claim 1).
The Applicant argues that the prior art does not teach adding a degradation tag to the second repressor. This argument is not persuasive because both Ohlendorf and Collins teach the addition of degradation tags to the repressors of their systems, where such tags could readily be added to dual repressor systems simply to achieve the desired effects of a practitioner to regulate and control the gene expression of the circuit. Ohlendorf already teaches the concept of tagging repressors in such genetic circuits, where furthermore Collins also teaches tagging the multiple repressors of their circuits with degradation tags; the present subject matter of claim 27 is therefore obvious.
Conclusion
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/D.C.R./Examiner, Art Unit 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635
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