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 .
Status of Claims
Receipt of Arguments/Remarks filed on 8/4/2025 is acknowledged. Claims 1 and 4-10 are pending. Claims 1 and 5-8 were amended. Claims 2 and 3 were canceled. Claims 4-10 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 5/22/2025.
Withdrawn Objections and Rejections
The amendment filed 8/4/2025 is sufficient to overcome the objection to claims 1 and 2. Claim 2 is canceled. Claim 1 is amended to define the acronym “RBS”.
The amendment filed 8/4/2025 is sufficient to overcome the rejection of claims 1-3 under 35 U.S.C. 112(b). Claim 1 has been amended to clarify what the sequence according to GenBank: AB248919.1 is referring to, and to clarify the options presented in previous claim 2, the limitations of which are now incorporated into claim 1. Claim 3 has been canceled.
The amendment filed 8/4/2025 is sufficient to overcome the rejection of claims 1-3 under 35 U.S.C. § 102/103. Claim 1 has been amended to recite a thermal switch vector that comprises the recited elements, rather than product-by-process limitations.
New and modified rejections necessitated by amendment
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 1 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites the limitation "the rhtC and the pycmt genes". There is insufficient antecedent basis for this limitation in the claim. There is no prior reference to rhtC and pycmt genes in claim 1 to indicate what “the rhtC and the pycmt genes” is referring to. It is suggested that the claim be amended to recite “an rhtC and a pycmt gene”.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim 1 is rejected under 35 U.S.C. 103 as being unpatentable over Zhou et al., Applied Biochemistry and Biotechnology; 178:324-37 in view of Lutz et al., Nucleic acids research; 25(6):1203-10; Zhao et al., Applied Microbiology and Biotechnology; 102:5505-18; Love et al., Gene; 176(1-2):49-53; Liu et al., Biotechnology and Bioengineering; 116(1):110-20; Kim et al., Journal of bacteriology; 192(20):5304-11; and Jiang et al., Applied microbiology and biotechnology; 97:5423-31.
Regarding claim 1, Zhou teaches a thermal switch vector (thermo-regulated genetic switch) for alanine production (Zhou “Abstract”). Zhou teaches a temperature sensitive circuit cIts-pR-p-L comprising the temperature sensitive repressor gene cIts and a tandem promoter pR-p-L (Zhou pg. 325 para. 3, Fig. 1). The nucleotide sequence GenBank: AB248919.1, or SEQ ID NO: 12, corresponds with the vector pPL451, as indicated in the instant specification Table 2. Zhou teaches the use of the vector pPL451 (Zhou pg. 326 “Strains”, Table 1). Zhou teaches a thermal switch circuit successively connecting cI-ts, pR-p-L, and a ribosome binding site/RBS (Zhou Fig. 1).
Zhou does not teach a rigorous circuit tetR-PLteto-1 wherein the tetR gene has the sequence shown in SEQ ID NO: 1, or a plasmid pFW001 with pMB1 replaced with replicon p15A and PJ23101 replaced with a thermal switch circuit. Zhou does not teach that the circuit successively connects cIts, the promoter pR-p-L, RBS, the repressor gene tetR, a multiple cloning site sequence MCS1, a terminator T7, the promoter PLteto-1, a multiple cloning site sequence MCS2 and a terminator T1 in series. Zhou does not teach an RBS according to SEQ ID NOs: 7 or 8. Zhou does not teach that rhtC or pycmt, which encodes a codon-optimized pyruvate carboxylase gene (instant specification pg. 8), are inserted in the thermal switch vector. Zhou does not teach that the vector contains the gene alaA labeled with an ssrA degraded peptide chain with a sequence according to SEQ ID NO: 10.
Regarding claim 1, Lutz teaches a rigorous circuit tetR-PLteto-1 for control of transcription units in E. coli (Lutz Table 1(a), Fig. 2). Lutz teaches a vector system comprising promoter/operator constructs, an RBS, two multiple cloning sites MCS1 and MCS2, an origin of replication p15A, and transcription terminators T1 and T0 (Lutz Fig. 2). Lutz teaches the sequence of tetR according to SEQ ID NO: 1 (see Search Results dated 4/1/2025, file “20250331_174608_us-17-746-130b-1.rge”, result 1). Lutz teaches a high strength RBS according to SEQ ID NO: 7 (see Search Results dated 4/1/2025, file “20250331_174608_us-17-746-130b-7.rge”, result 33).
Zhao teaches the vector pFW01, constructed from pF2, by inserting the PJ23101 promoter (Zhao pg. 5508 “Construction of different plasmids”). pF2 contains the replicon pMB1 (see Addgene, plasmid map). Zhao additionally teaches that the gene rhtC, which is one of the most efficient exporters of threonine, is expressed in an E. coli strain for enhanced threonine production (Zhao “Abstract”, pg. 5515 “Comparison of L-threonine production in different E. coli TWF006 derivatives”).
Love teaches vectors with tandem pR and pL promoters upstream of an extensive multiple cloning site (MCS) for insertion of genes and direct expression of the clts857 gene, enabling their use in any Escherichia coli host strain for thermal induction of gene overexpression (Love “Abstract”). Love teaches the vector pPL451. Love additionally teaches an RBS corresponding with SEQ ID NO: 8 (see Search Results dated 4/1/2025, file “20250331_174608_us-17-746-130b-8.rge”, result 1).
Liu teaches expression of pyruvate carboxylase, pyc, in E. coli for the production of threonine (Liu pg. 113 Section 3.1). Liu additionally teaches codon optimization of genes for expression in the recombinant E. coli strain (Liu pg. 113 Section 2.3).
Kim teaches that alaA, also called yfbQ, is a major enzyme in alanine synthesis in E. coli (Kim “Abstract”). alaA/yfbQ is a transaminase that converts pyruvate to alanine. Jiang teaches a temperature sensitive cI857-PL vector system for protein expression in E. coli (Jiang “Abstract”).
Jiang teaches the use of a standard ssrA tag (AANDENYALAA) at the 3’ terminal end of the lacI gene (Jiang pg. 5426 “System design and construction”).
It would have been obvious to a skilled artisan, before the effective filing date, to combine the teachings of these references, arriving at the invention as instantly claimed. All of these references are directed to engineered bacterial strains to control gene expression. It would have been obvious to combine the teachings of Zhou and Lutz, incorporating a temperature sensitive circuit as taught by Zhou and a rigorous circuit as taught by Lutz into the same vector. It would additionally be obvious to use the plasmid as taught by Zhao, which includes the PJ23101 promoter and the pMB1 replicon, to construct the vector, as this plasmid is used for L-threonine production, similar to the plasmid used for alanine production as taught by Zhou. It would have been obvious to a skilled artisan that the pMB1 replicon in the vector as taught by Zhao could be substituted with a medium copy number p15A replicon, as Lutz teaches the use of several alternative origins of replication including p15A and ColE1, which is similar to pMB1 (Lutz Fig. 2).
Additionally, it would have been obvious that the high strength RBS taught by Lutz or the low strength RBS taught by Love could both be used in a vector as taught by Zhou. Zhou teaches cI-ts, pR-p-L, and an RBS connected in series, and it would have been obvious to a skilled artisan to additionally connect the repressor, MCS1, terminator, PLteto-1, MCS2, and another terminator in series, as these elements are all taught by Lutz. While Lutz does not expressly teach the T7 terminator, it would be obvious to a skilled artisan to substitute one known terminator T0, for another, T7, with an expectation of similar results.
It would have further been obvious to a skilled artisan to combine the teachings of Zhou, Lutz, Zhao, Love and Liu. The vectors taught by Zhou and Zhao are both used for amino acid production. It would have been obvious to a skilled artisan that the rhtC and pyc genes, which are known to be involved in threonine production, could be incorporated into a thermal switch vector as taught by Zhou, Lutz, Zhao, and Love. It would have additionally been obvious to codon-optimize pyc, as Liu teaches codon optimization of some genes for recombinant expression, and this is a known technique in the art that would be performed by a skilled artisan during routine optimization. It would have been obvious to a skilled artisan that the rhtC and pyc genes could be either co-expressed by being inserted successively (pFT26rp) or could be expressed on different vectors, i.e. pFT24r or pFT24p.
It would have been obvious to a skilled artisan to combine the teachings of Zhou, Lutz, Zhao, Love, and Liu with the teachings of Kim and Jiang, arriving at the vector as instantly claimed. alaA is involved in amino acid production as taught by Kim, and it would have been obvious to a skilled artisan that this gene could be incorporated into a thermal switch vector for amino acid production. Jiang teaches the use of a standard ssrA tag for protein degradation used in a thermal switch vector system, so it would additionally have been obvious to incorporate a standard ssrA tag according to instant SEQ ID NO: 10 into the thermal switch vector.
A person of ordinary skill in the art would have been motivated to combine these teachings because genetic switches such as the temperature sensitive switch and the rigorous circuit are useful tools for genetic engineering. Lutz teaches that the temperature sensitive cI857 vector system has the downside of no quantitative control of time and additional pleiotropic effects (Lutz pg. 1203 “Introduction”). The rigorous circuit as taught by Lutz allows for quantitative and independent control over transcriptional units and using this system of tight control in combination with a temperature sensitive circuit as taught by Zhou would be considered an improvement upon the system of Zhou (Lutz “Introduction”).
A person of ordinary skill in the art would have further been motivated to co-express the rhtC and pyc genes because these genes are used in recombinant strains for threonine production, so it would be desirable to express these genes in a thermal switch vector used for threonine production.
A person of ordinary skill in the art would have additionally been motivated to combine these teachings and incorporate alaA because alaA is involved in conversion of pyruvate to alanine. Zhao teaches metabolic engineering techniques to enhance the production of threonine, involving modified expression of genes within the L-threonine biosynthetic pathway to control carbon flux distribution (Zhao Fig. 1). It would therefore be considered advantageous for a skilled artisan to control the expression of alaA if using a thermal switch vector for threonine production, as reduced alaA activity would allow more pyruvate availability for threonine biosynthesis, rather than pyruvate being converted to alanine. A standard ssrA degradation tag, including a tag with the sequence of SEQ ID NO: 10, could be used for such a purpose, as taught by Jiang.
A skilled artisan would have a reasonable expectation of success in making this combination to achieve the predictable outcome of a thermal switch vector as instantly claimed, as each of the elements of this vector are known in the art for gene regulation in recombinant E. coli and could be combined into a single vector using techniques known to those skilled in the art. Additionally, a thermal switch vector for amino acid production is known in the art as taught by Zhou, and expression of amino acid biosynthesis genes such rhtC and pyc is an established technique for creating recombinant strains with enhanced amino acid production. Finally, a skilled artisan would have a reasonable expectation of success in making this combination because it is known in the art that modification of enzymes in an amino acid biosynthetic pathway can lead to enhanced or reduced production of specific amino acids, and modification of alaA expression is known to influence alanine production as taught by Kim.
Response to Arguments
Applicant’s arguments, filed 8/4/2025, have been fully considered, and in light of amendments to the claims, the rejection of claims 1-3 under 35 U.S.C. § 102/103 has been withdrawn.
Applicant’s arguments, filed 8/4/2025, have been fully considered, and in light of amendments to the claims, the rejection of claim 1 under 35 U.S.C. § 103 in view of Zhou et al, Lutz et al., Zhao et al., and Love et al. has been withdrawn. The rejection of claim 2 under 35 U.S.C. § 103 in view of Zhou et al, Lutz et al., Zhao et al., Love et al., and Liu et al. has been withdrawn. The rejection of claim 3 under 35 U.S.C. § 103 in view of Zhou et al, Lutz et al., Zhao et al., Love et al., Liu et al, Kim et al., and Jiang et al. has been withdrawn.
However, upon further consideration, new grounds of rejection of claim 1 are made under 35 U.S.C. § 103 as set forth above. Given these new grounds of rejection, the arguments presented regarding claims 1-3 rejected under 35 U.S.C. § 103 are moot.
Applicant argues that amended claim 1 now incorporates elements previously recited in claims 2 and 3, and these elements are not taught by Zhou et al, Lutz et al., Zhao et al., and Love et al. The newly applied rejection under 35 U.S.C. § 103 incorporates the teachings of Liu et al, Kim et al., and Jiang et al., which cure these deficiencies.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/EMILY F EIX/Examiner, Art Unit 1653
/JENNIFER M.H. TICHY/Primary Examiner, Art Unit 1653
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