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 .
Application Status
This action is written in response to applicant’s correspondence received on 4/17/2024. Claims 1-20 and 22-26 are pending. Claim 21 has been cancelled. All pending claims are currently under examination.
Any rejection or objection not reiterated herein has been overcome by amendment. Applicant’s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow. This Office Action is Final.
Declaration Under 37 CFR 1.132
The declaration under 37 CFR 1.132 filed 9/5/2023 is insufficient to overcome the rejection of claims 1-20 and 22-26 based upon Ohlendorf (Ohlendorf R et al. J Mol Biol. 2014 Jan 24;426(2):500), Marbach (Marbach A et al. J Biotechnol. 2012 Jan;157(1):82-8), Falb (US Patent US 20170216370 A1, 2017), Lewis (Lewis M. The lac repressor. C R Biol. 2005 Jun;328(6):521-48), Zucca (Zucca S et al. J Biol Eng. 2013 May 10;7(1):12), Martin 1(Martin VJ et al. Nat Biotechnol. 2003 Jul;21(7):796-802), Martin 2 (Martin VJ et al. Biotechnol Bioeng. 2001 Dec 5;75(5):497-503), Jayaraman (Jayaraman P et al. Nucleic Acids Res. 2016 Aug 19;44(14):6994-7005), and Murray (WO-2016134069-A1) under 35 U.S.C 103 as set forth in this Office Action for the reasons discussed below. Furthermore, arguments presented in the declaration were addressed in the Final Office action mailed on 9/27/2023. These arguments regarding the declaration are still valid because the claims have not been substantially amended.
Claim Rejections - 35 USC § 103 - Maintained
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 for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-7, 10-14, 16-18, and 20, 23, and 26 are 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) and Falb (US Patent US 20170216370 A1, 2017). The rejection of claim 5 is further evidenced by Lewis (Lewis M. The lac repressor. C R Biol. 2005 Jun;328(6):521-48).
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.
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 (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 (third sequence). 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).
In addition, Falb, a US patent granted 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 Marbach (Abstract and Title) and 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, Ohlendorf, and Marbach overlap in scope in the sense that they are all bacterial engineered systems which teach the use of the same repressor and induction systems.
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 and Falb to arrive at the present invention because incorporating the lac repressor into pDawn is the application of a known technique (light-induction using pDawn) to a known product (the ubiquitous lac operon bacterial systems used in the industry) ready for improvement to yield predictable results. Ohlendorf draws a direct comparison of light-induced expression systems to IPTG-induced expression systems and states that it would be better to use the light-controlled pDawn system to induce gene expression in place of using IPTG (Discussion, first paragraph, lines 14-18), and it was known that IPTG controls the induction of the lac operon (above). Thus, Ohlendorf teaches that chemical induction using IPTG was ready for improvement by using the pDawn vector. A person of ordinary skill in the art would recognize that in order to build a light-controlled expression system to replace IPTG induction, a practitioner could simply incorporate the elements of the lac operon system with the pDawn system. As Marbach discloses, IPTG is a chemical inducer which inhibits the ability of a repressor (LacI) to act on its target DNA sequence. In order to follow the suggestion of Ohlendorf, to use the light-inducible pDawn system to replace chemical IPTG induction, one could simply place the lacI repressor downstream of the pR promoter of the pDawn system. As discussed above, the pDawn system has a multiple cloning site (MCS) in this region, which means that it is inherently designed to be modified by a practitioner at this location. Additionally, Falb teaches two-repressor regulatory genetic circuits in the field of bacterial engineering (paragraph 196), and therefore teaches that two-repressor systems were known in the field of bacterial engineering. Modifying the pDawn system of Ohlendorf with the lac operon taught by Marbach is therefore merely a combination of two known systems, whose combination produces predictable results, where such a combination can further be characterized as the application of the known technique of using light to induce expression of a gene (Ohlendorf) to a known product (the lac operon) ready for improvement to yield predictable results because Ohlendorf taught that using light-induction is an improvement over IPTG-induction. Each of these two systems (pDawn and the lac operon) contains a repressor, and as taught by Falb, genetic circuits containing two repressors are already known in the art.
Furthermore, as taught by Marbach, the lac operon is a ubiquitous system in protein expression. A practitioner would therefore understand that incorporating elements of the lac operon (for instance, the lac repressor) into the pDawn system would be a logical way to replace IPTG-inducible systems: a practitioner would understand that it would be simpler to create a single pDawn plasmid containing the lac repressor than it would be to generate thousands of new plasmids with individual genes of interest using the pDawn system. Given that it was known that the lac operon is used ubiquitously in the industry, a practitioner in the art would understand and be motivated to incorporate the molecule which controls repression of the lac operator (i.e., the lacI repressor) into the pDawn system in order to follow the suggestion of Ohlendorf to replace IPTG with light as an inducer at the industry level. Sufficient motive is supplied by Ohlendorf to create such a construct, as Ohlendorf states that “light induction is noninvasive, which reduces risk of contamination, and cos-effective [sic],” Discussion, first paragraph. Additionally, and importantly, as discussed above, Marbach teaches the ubiquity of the lac operon in the industry. A practitioner would therefore be motivated to target the regulation of the lac operon by using the pDawn system when trying to convert chemically-induced systems to light-induced systems, as suggested by Ohlendorf, owing to the widespread use in the lac operon in the industry. The practitioner would be motivated to ensure that the pDawn system would be compatible with the lac operon because Marbach teaches that the lac operon is a ubiquitous genetic circuit in bacterial protein expression systems. Furthermore, the combination of Ohlendorf with the teachings of Marbach yields predictable results in that a repressor (LacI) will still function to repress its target gene in the disclosed system, thereby controlling expression of the target gene in a light-controllable manner.
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.
Regarding claims 2 and 3, Ohlendorf teaches that pDawn is controlled by the two-component system YF1/FixJ (Results, line 5).
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 pDawn system used by Ohlendorf modified by the teachings of Marbach and expressed in a microorganism arrives at the system described in claim 16. Rationales and motives to create such a system are discussed above for claims 1-14. Furthermore, bacterial expression systems using two repressors were known in the art, as evidenced by Falb, 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.
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.
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) and Falb (US Patent US 20170216370 A1, 2017) 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, and Falb teaches the system of claim 1, wherein sequences of the system are found in a plasmid.
Ohlendorf, Marbach, and Falb 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, and Falb 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, 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, and Falb teach the elements of claim 1, as discussed above. Ohlendorf, Marbach, and Falb 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 and Marbach 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, as applied to claim 21 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, and Falb arrive at the method described in claim 16 and further refined in claim 21, as discussed above. However, Ohlendorf, Marbach, and Falb 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, and Falb 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, and Falb with a method taught by Jayaraman with predictable results.
Claims 24-25 are 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) and Falb (US Patent US 20170216370 A1, 2017). as applied to claim 1 above, and further in view of Murray (WO-2016134069-A1).
Regarding claims 24 and 25, as discussed above, a combination of Ohlendorf, Marbach, and Falb yields the two-repressor light-controllable system of claim 1. Additionally, 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.
Ohlendorf, Marbach, and Falb do not teach or suggest that the first nucleotide sequence includes an additional sequence controllable by the second repressor downstream from the first promoter.
Murray is a patent document that teaches methods for designing genetic circuits and also teaches an embodiment of a genetic circuit that is a two-repressor system. In paragraph 118, final sentence, Murray teaches that, with respect to the genetic circuits they describe, “in some embodiments, molecular components can be connected in one or more motifs to achieve desired functionality at a higher-order of complexity,” which suggests that a practitioner of ordinary skill in the art can choose a desired functionality and the desired level of complexity for a genetic circuit, and further that how the genetic circuit would function would be predictable. Murray teaches the following regarding an embodiment of their invention: “a genetic circuit can be a genetic toggle switch that comprises two repressors mutually repressing each other. For example, Figure 3A shows a genetic toggle switch having two repressors TetR and LacI which mutually repress each other” paragraph 119 and Figure 3A. Murray therefore teaches two-repressor systems capable of repressing each other, wherein the second repressor is LacI. Figure 3A of Murray illustrates this negative feedback loop.
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 two-repressor light-inducible system taught by Ohlendorf, Marbach, and Falb to include a negative feedback loop as taught by Murray, so that the second repressor is capable of controlling the first repressor by binding to the first promoter as illustrated in Figure 3A of Murray. The combination of these elements is simply a combination of prior art elements according to known methods to yield predictable results. Murray teaches that the design of genetic circuits have predictable results, and can be used for a desired function selected by a practitioner of ordinary skill. Each of the elements of the present invention were known, and the combination of said elements is equal to the sum of its parts. Additionally, the functions of each of the known elements are maintained (i.e., repressors function as repressors, light-inducible systems function as light-inducible systems etc.).
Regarding claim 25, as discussed above, Ohlendorf used degradation tags in their system for the repressor cI, and using the degradation tag improved the response dynamics of their system. Therefore, it would be obvious to include a degradation tag on the second repressor because, as taught by Ohlendorf, by so doing a practitioner would improve the response dynamics of a light-controllable system.
Response to Arguments
Applicant's arguments filed 4/17/2024 have been fully considered but they are not persuasive.
The Applicant argues that the radical change that optogenetic control provides compared with chemical induction to control a bioreactor has not been considered. This argument is not found to be persuasive. It is understood that using light to control protein expression in a bioreactor is a departure from conventional chemical induction methods using, for instance, IPTG. However, the Applicants have not originated this concept. As taught by Ohlendorf:
“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,” (page 539, left column, first paragraph).
Ohlendorf therefore teaches that light induction can supersede conventional chemical induction methods using IPTG (page 539, left column, first paragraph). Furthermore, Ohlendorf reduces to practice induction/expression of recombinant proteins using light instead of chemicals, teaches that the yield of such systems rivals that of IPTG induction, and also teaches that light-induction using their pDawn system should be used in place of conventional IPTG-induction for several advantageous reasons (page 538, left column, page 539, and Abstract). It is therefore accepted that light-induction to express recombinant proteins is a departure from chemical induction methods, as taught by Ohlendorf who teaches that light-induction should supersede chemical induction (page 539, left column, page 538, left column, and Abstract). However, this “radical” change concerning the control of the expression of recombinant expression systems using light as opposed to chemical induction is not an idea that is original to the present Applicant, as taught by Ohlendorf (page 538-539, Abstract).
The Applicant argues that it does not make sense to control the lacI repressor using the system of Ohlendorf. This argument is not persuasive. Marbach teaches that IPTG-inducible systems using the lac operon and lacI repressor are ubiquitous in the industry (Abstract and Introduction). A practitioner would therefore be strongly motivated to incorporate the lac repressor into the system taught by Ohlendorf because such a modification would make the vector of Ohlendorf compatible with the industry-wide, ubiquitously-known lac system currently used. Thus, the incorporation of the lac repressor into the pDawn vector of Ohlendorf is the application of a known technique (light-induction using pDawn) to a known product (the lac operon and its circuitry) ready for improvement to yield predictable results. Ohlendorf teaches that IPTG-inducible systems are ready for improvement by using the light-induction system that they have created (pages 538-539, Abstract). The practitioner would be additionally motivated to use the lac repressor in combination with Ohlendorf’s pDawn vector because this repressor is widely used in in the industry (Marbach, Abstract and Introduction), and is therefore an obvious target for light-inducible systems. In light of the state of the industry, it makes sense to clone the ubiquitous lac repressor into the pDawn vector of Ohlendorf, so that pDawn would be compatible with the most widely used genetic circuit in the industry (Marbach, Abstract and Introduction). The creation of such a plasmid would make pDawn compatible with the current protein expression industry.
The argument that a practitioner would simply clone a gene of interest into the pDawn vector, and not the lac repressor, does not take into consideration the state of the industry: a practitioner of ordinary skill in the art would understand that IPTG-inducible lac systems are ubiquitous in the industry, and would therefore be motivated to incorporate the lac repressor into the pDawn vector of Ohlendorf in order to adapt light-inducible systems taught by Ohlendorf to be used with the current state of the industry, which relies ubiquitously on the lac operon/lac repressor.
Furthermore, given that a practitioner would be motivated to clone the lac repressor into the pDawn vector of Ohlendorf, this would inherently make a two-repressor system, regardless of the teachings of Falb. Thus, the motive to make a two-repressor system is a combination of Ohlendorf and cloning the lac repressor of Marbach into Ohlendorf’s pDawn vector. Falb teaches that such repressor systems are possible, and further that such systems can also use the same inducible systems taught by Marbach and Ohlendorf (paragraph 264). A practitioner would be motivated to include the repressor taught by Marbach with the pDawn system of Ohlendorf, independently of the teachings of Falb. Falb simply teaches that such a combination would reasonably be expected to work because Falb teaches two-repressor systems using the same components of Marbach and Ohlendorf (paragraph 264).
The Applicant argues that Ohlendorf’s teachings are inoperable in Falb. This argument is not persuasive. The teachings of Falb are discussed to modify Ohlendorf; Ohlendorf is not modifying Falb. Therefore, the fact that optogenetic control of circuits (Ohlendorf) in the teachings of Falb is inoperable is irrelevant because the teachings of Falb modify the teachings of Ohlendorf, not the other way around. In short, the teachings of Ohlendorf and Marbach render obvious two-repressor systems (pDawn of Ohlendorf with the lacI repressor of Marbach), and Falb teaches that such systems are known to work in the art. The combination of the prior art therefore has a reasonable expectation of success.
In addition, Applicants have provided only arguments of counsel, and arguments of counsel cannot take the place of factually supported objective evidence. See, e.g., In re Huang, 100 F.3d 135,139-40, 40 USPQ2d 1685, 1689 (Fed. Cir. 1996); In re De Blauwe, 736 F.2d 699, 705, 222 USPQ 191, 196 (Fed. Cir. 1984).
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
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/D.C.R./Examiner, Art Unit 1636
/NANCY J LEITH/Primary Examiner, Art Unit 1636
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