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 .
The examiner of your application in the PTO has changed. To aid in correlating any papers for this application, all further correspondence regarding this application should be directed to Brian Whiteman, Art Unit 1636.
Election/Restrictions
Claims 18-27 remain withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.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 12/12/24.
Specification
The amendment to the specification filed on 6/24/25 has been entered.
Claim Objections
Claim 1 is objected to because of the following informalities: the term “a.” and “b.” should be replaced with a) and b) because only one period should be at the end of the claim unless the period is for an abbreviation of a term. See MPEP 608.01(m). Appropriate correction is required.
Response to Arguments
Applicant’s arguments, see pages 13-25, filed 6/24/25, with respect to the rejection(s) of claim(s) 1-16 under 102 and 103 rejections based on Martin (US 201002161960) have been fully considered and are persuasive. Therefore, the rejection has been withdrawn in view of the amendment to claim 1 to require a host cell comprising laczΔ15 in a lac operon and the cassette now requires a LacZYA promoter or a OmpF promoter operably linked to the a nucleic acid sequence encoding the amino terminal fragment of β-galactosidase, wherein the amino acid fragment comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 1. However, a new rejection(s) is required in view of the amendment to the claims.
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.
Claims 1, 3-5, 8, 12, and 15 are rejected under 35 U.S.C. 103 as being unpatentable over Slilaty et al. (US 6127171, of record).
Slilaty claims: A method of using vector for cloning a DNA molecule, wherein the vector comprises at least one promoter operably linked to a modified Escherichia coli lacZ coding sequence encoding an α-peptide and containing at least one cloning site cleavable by a restriction enzyme wherein the modified lacZ coding sequence is made by introducing at least one cloning site within a lacZ coding sequence downstream of and including the codon corresponding to the codon designated as codon 8 in the native E. coli lacZ coding sequence, wherein the method comprises:
(a) cloning a DNA molecule into the at least one cloning site in the modified lacZ coding sequence encoding an α-peptide to form recombinant vectors;
(b) introducing the recombinant vectors into host cells, wherein the host cells require α-complementation to produce β-galactosidase activity;
(c) growing the host cells in the presence of a chromogenic substrate or indicator for β-galactosidase activity; and (d) screening for indicia of lac operon marker inactivation. See claims 1-29 in columns 26-28.
Slilaty also teaches that "lacZα is a gene fragment, comprising the proximal portion of the Escherichia coli lacZ gene, which encodes approximately 60 of the amino terminal amino acids of the ß- galactosidase polypeptide chain. The encoded product, the "α-peptide", complements the defective activity of the gene product of lacZM15, also known as lacZΔ15 deletion (column 1, last paragraph). Slilaty also teaches that the lacZa (α-peptide) is operably linked to a promoter (Abstract; column 3, last paragraph). Further, Slilaty teaches an amino-terminal fragment of ß-galactosidase (SEQ ID NO: 1) that comprises the amino acid sequence of claimed SEQ ID NO: 1 (see sequence alignment below: Qy = sequence from instant claims, Db = sequence from Slilaty).
Qy
1MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWR 60
Db
1MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWR 60
See also columns 9-20 for the working examples. Example 6 (columns 17-20) discloses using a vector comprising a modified lacZ coding sequence having a DNA molecule into a restriction enzyme site in the lacZ coding region or in a vector sequence which is upstream of a region of the modified lacZ coding sequence that corresponds to DNA sequence encoding amino acids 8 to 38 of β-galactosidase as shown in SEQ ID NO: 1.
Slilaty further contemplates:
Various bacterial, phage, or plasmid promoters known in the art from which a high level of transcription has been observed in a host cell system such as E. coli include, but are not limited to, the lac promoter, trp promoter, tac promoter, recA promoter, ribosomal RNA promoter, the P.sub.R and P.sub.L promoters, T7 promoter, SP6 promoter, lacUV5, ompF, bla, and lpp. Column 8, lines 8-25.
Slilaty does not specifically teach a method comprising contacting a cell comprising a lacZΔ15 deletion in a lac operon with a nucleic acid construct comprising an expression cassette comprising a LacZYA promoter or an OmpF promoter operably linked to a nucleic acid encoding the amino terminal fragment of β-galactosidase, wherein the amino terminal fragment comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 1; and growing the host cell under conditions for the nucleic acid construct to be maintained in the cell.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified a plasmid vector comprising lacZ α-peptide of Slilaty comprising a sequence that is 95% identity to amino acid sequence of instant SEQ ID NO: 1 with any promoter that would have a high level of transcription in E. coli, such as an ompF promoter as suggested by Slilaty, as it would have merely amounted to a simple substitution of one known promoter for high level transcription in E. coli into the plasmid vector to yield predictable results. See MPEP 2143(I)A, B, C, and G. A person of ordinary skill in the art would have been motivated to try modifications upstream or downstream of SEQ ID NO: 1 in the vector or a vector comprising the ompF promoter operably linked to a DNA sequence encoding the alpha-peptide comprising SEQ ID NO: 1, wherein the resultant peptide is capable of complementation with a defective beta-galactosidase molecule in the cell as a control or to compare color selection mechanism to the plasmid vectors made in the working examples of Slilaty (columns 3-4 and 19-20). One would have a reasonable expectation of using the method steps of the claimed method with success in view of the teaching of Slilaty. Also, Slilaty teach origin of replications (ori) are well-known in the prior art (columns 4, 9-13 and claims 13) and uses a plasmid vector comprising an ori. One of ordinary skill in the art would have been motivated to add a replication origin to the plasmid vector for replication of the vector. Slilaty teach adding a colE1 replication of origin to the plasmid vector, which a person of ordinary skill in the art would possess the knowledge is known as a high-copy origin of replication (column 9 and claim 13).
Therefore, the invention as a whole would have been prima facie obvious to one ordinary skill in the art before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains.
Response to Arguments
Applicant's arguments filed 6/24/25 have been fully considered but they are not persuasive.
NOTE: even though the 103 rejection is new because of the amendment to claim 1, the response to arguments will be addressed because Slilaty and what was known in the prior art of record was cited in the arguments.
In response to applicant’s argument that the cited prior art do not teach a nucleic acid sequence operably linked to a LacZYA or a OmpF promoter an amino terminal fragment of beta-galactosidase comprising an amino acid sequence with at least 95% identity to SEQ ID NO: 1, the argument is not found persuasive because Slilaty contemplates and provides motivation for using a OmpF promoter in a plasmid vector comprising a nucleotide construct encoding a sequence that is at least 95% identical to SEQ ID NO: 1. Column 8, lines 8-25. Slilaty provides motivation for using the promoter because it is considered a high-level transcription promoter.
Applicant argues the prior art, including Slilaty, do not provide a skilled artisan with any predictability or expectation of success at arriving at the claimed method because the prior art (Martin, US 20100216190) used an alpha complementation assay using the E. coli XL-1 blue stain and the pUC19 expression vector and the pU19 was unable to complement the LacZdeltaM15 of the TOP 10 cells and therefore a skilled artisan would not be able to use any nucleic acid construct comprising the claimed isolated beta-galactosidase expression cassette with any host cell comprising a LacZdeltaM15 to perform an alpha-complementation assay based on teaching of Martin.
Applicant’s argument is not found persuasive because Martin is not cited in a 103 rejection. Slilaty makes obvious using a OmpF promoter in a plasmid vector comprising an expression cassette comprising a nucleic acid encoding an amino acid sequence with at least 95% identity to SEQ ID NO: 1 and introducing the vector into competent host cells; growing the host cells in the presence of a chromogenic substrate cleavable by .beta.-galactosidase; and screening for indicia of lac operon marker inactivation. Slilaty teaches the high accuracy of color selection afforded by the modified lacZ.alpha. coding sequence of the present invention provides an opportunity at performing gap-free shotgun sequencing and development of ordered genomic libraries. The method made obvious by Slilaty does not embrace using pUC19 plasmids in an alpha complementation assay in TOP10 cells. The claimed method is broader than using pUC19 plasmids in an alpha complementation assay and embraces the method made obvious by Slilaty.
Applicant further argues that Example 2 of the present disclosure shows that using expression cassettes with medium and strong promoters the engineered LacZ-alpha expression cassettes are able to complement the LacZdeltaM15 of TOP10 cells. Furthermore, Example 5 of the present disclosure demonstrates that the claimed vector in different types of host cells.
Applicant’s argument is not found persuasive because Slilaty makes obvious the claimed method and example 2 of the as-filed specification further confirms that the vector made obvious by Slilaty would work in host cells. In addition, the method made obvious by Slilaty does not embrace using pUC19 plasmids in an alpha complementation assay in TOP10 cells.
In response to applicant’s argument that Example 4 of the present disclosure demonstrates that the isolated vector of the present invention is able to complement LacZdeltaM15 cells even when a large insert was cloned into the isolated vector, the argument is not found persuasive because the claimed method is not directed or embrace using a plasmid vector with a large insert. The method made obvious by Slilaty does not appear to embrace using pUC19 plasmids in an alpha complementation assay in host cells. In addition, Slilaty discloses a method of using a plasmid vector comprising a nucleic acid construct having a sequence of SEQ ID NO: 1 could be used with a large insert could be used in a method. See column 14 of Slilaty which teaches, “The additional advantage of the phage vector according to the present invention is that the it would be add stability of DNA inserts, as compared to currently used M13 phage”.
Applicant further argues that Example 2 further demonstrates that the isolated vectors are efficiently maintained under non-selective conditions even after several cycles of 1:50,000 dilutions and Page 23, lines 26-28 of the as-filed specification notes that pUC vectors are lost from cells at a high rate in the absence of selection. The isolated vectors of the present invention are efficiently maintained in LB liquid cultures absence of selective pressure. Example 4 further demonstrates that the isolated vector with the cloned insert is efficiently maintained even without selection. Thus, a skilled artisan would not be able to predict that a nucleic acid construct comprising an isolated beta-galactosidase expression cassette comprising a nucleic acid encoding the amino terminal fragment of beta-galactosidase would be maintained under non-selective conditions from the teaching of Martin, Addgene1 and Slilaty.
Applicant’s argument is not found persuasive because Martin and Addgene1 are not cited in the new 103 rejection. Slilaty makes obvious using a OmpF promoter in a plasmid vector comprising an expression cassette comprising a nucleic acid encoding an amino acid sequence with at least 95% identity to SEQ ID NO: 1 and introducing the vector into competent host cells; growing the host cells in the presence of a chromogenic substrate cleavable by .beta.-galactosidase; and screening for indicia of lac operon marker inactivation. Slilaty teaches that the high accuracy of color selection afforded by the modified lacZ.alpha. coding sequence of the present invention provides an opportunity at performing gap-free shotgun sequencing and development of ordered genomic libraries and this provides motivation to make a method that would read on the claimed invention. The method made obvious by Slilaty does not appear to embrace using pUC19 plasmids in an alpha complementation assay in host cells.
Claims 6-7 are rejected under 35 U.S.C. 103 as being unpatentable over Slilaty (supra) as applied to claims 1, 3, 4, 5, 8, 12, and 15 above, and further in view Ye (US 20160194660).
Slilaty teaches making a plasmid vector comprising an origin of replication, but does not specifically teach the plasmid vector comprising a high copy replication of origin set forth in claim 6.
However, Ye teaches making a plasmid comprising pUC19 origin of replication comprising SEQ ID NO: 19. See pages 4, 7-8 and 24-28. SEQ ID NO: 19 is 100% identical to instant SEQ ID NO: 13 in claim 7. See attached OA appendix for alignment.
SEQ ID NO: 19 in Ye is 100% identical to instant SEQ ID NO:13, even though the term in the instant claims for SEQ ID NO: 13 is pUC57, the origin of replication in Ye would inherently read on the term recited in claims 5-7.
It would have been prima facie obvious to a person of ordinary skill in the art before the time of the effective filing date to combine the teaching of Slilaty taken with Ye as a simple substitution to use a known replication of origin (instant SEQ ID NO: 19) to the plasmid vector made obvious by Slilaty, namely to arrive at the claimed invention. Since the sequence taught by Ye is 100% identical to instant SEQ ID NO: 19, it would read on a high-copy replication of origin (pUC57 replication origin).
Therefore the invention as a whole would have been prima facie obvious to one ordinary skill in the art before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains.
Claims 9-10 are rejected under 35 U.S.C. 103 as being unpatentable over Slilaty (supra) as applied to claims 1, 3-5, 8, 12, and 15 above, and further in view Cranenburgh et al. (WO 2012001352, of record).
Slilaty does not specifically teach a dimer resolution further comprising a nucleic acid encoding a site-specific recombinase.
Cranenburgh teaches plasmids containing selectable markers, site-specific recombinase target sites, including the cer site, and recombinases, including XerC and XerD (Abstract) page 1; paragraphs 3 and 4, page 7).
Cranenburgh teaches that when site specific recombinases, such as the Xer recombinases, are not naturally present in the host cell, genes encoding the recombinases may be introduced on an extrachromosomal element, and that this would allow for the expression of site-specific recombinases capable of acting upon the site-specific recombinase target site (Introduction of recombinase, lines 5-30, page 8).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Slilaty by incorporating the teachings of Cranenburgh to include the XerC/D site-specifc recombinases on the plasmid containing a cer locus. Doing so would allow expression of the XerC/D recombinases and recombination at the cer site in host cells that don't naturally express XerC/D, as recognized by Cranenburgh. Further, as the teachings of Cranenburgh relate to the cer recombinase recognition site and the XerC and XerD recombinases, there is a reasonable expectation that XerC and XerD site-specific recombinases could be successfully applied to the plasmid vectors of Slilaty.
Response to Arguments
Applicant's arguments filed 6/24/25 have been fully considered but they are not persuasive because the only arguments against this additional reference is directed at Slilaty. The argument is not found persuasive for the reasons set forth above in response to Slilaty and incorporated herein.
Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Slilaty (supra) as applied to claims 1, 3-5, 8, 12, and 15 above, and further in view Blakely et al. (Cell 75, pages 351-361, 1993, of record).
Slilaty teaches various bacterial, phage, or plasmid promoters, including ompF have a high level of transcription in a host cell system such as E. coli
Slilaty does not specifically teach wherein the host cell comprises a nucleic acid encoding a site-specific recombinase.
Blakely teaches that the E. coli chromosome encodes 2 recombinases, XerC and XerD, for site specific recombination at cer (Title and Summary, page 351; Introduction, second and third paragraphs, page 351).
Therefore, Slilaty inherently teaches that the E. coli host cell comprises a nucleic acid encoding a site-specific recombinase.
It would have been prima facie obvious to a person of ordinary skill in the art before the time of the effective filing date that when using E. coli as the host cell, the host cell would inherently read on the limitations of instant claim 11.
Therefore the invention as a whole would have been prima facie obvious to one ordinary skill in the art before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains.
Response to Arguments
Applicant's arguments filed 6/24/25 have been fully considered but they are not persuasive because the only arguments against this additional reference is directed at Slilaty. The argument is not found persuasive for the reasons set forth above in response to Slilaty and incorporated herein.
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Slilaty (supra) as applied to claims 1, 3-5, 8, 12, and 15 above, and further in view of ¡GEM (Part:BBa_K2847001, https://parts.igem.org/Part:BBa_K2847001, 10/09/2018, [retrieved 03/19/2025]).
Slilaty does not specifically teach the ColEi dimer comprising instant SEQ ID NO: 20.
¡GEM teaches the inverted cer sequence from the plasmid ColE1, that when reversed, comprises a sequence with 100% identity to SEQ ID NO: 20 from the instant application (see sequence alignment in non-final rejection mailed on 3/28/25). iGEM also teaches that "Cer plays role as part from the cer-xer system that converts dimers by recombination back to monomers to stabilize the plasmid."
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Slilaty by incorporating the teachings of iGEM to include the exact sequence of cer on the plasmid containing the cer locus. Doing so would improve plasmid stability through recombination of plasmid dimers back to monomers, as recognized by iGEM. Further, as the teachings of both Slilaty and iGEM relate to plasmids and the cer-xer recombination system, there is a reasonable expectation that cer sequence could be successfully applied to the plasmid vectors of Slilaty.
Therefore the invention as a whole would have been prima facie obvious to one ordinary skill in the art before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains.
Response to Arguments
Applicant's arguments filed 6/24/25 have been fully considered but they are not persuasive because the only arguments against this additional reference is directed at Slilaty. The argument is not found persuasive for the reasons set forth above in response to Slilaty and incorporated herein.
Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Slilaty (supra) as applied to claims 1, 3-5, 8, 12, and 15 above, and further in view of Lu et al. (Mol. Ther. 20, 2111-9, 2012, cited on an IDS).
Slilaty does not specifically teach the plasmid vector is less than 1.5 kilobases in size.
Lu et al. teach that extragenic DNA ≥ 1 kb in plasmid vectors results in silencing of transgene expression (Abstract, page 2111; Discussion, first paragraph, page 2117). Lu teaches that the silencing is due to the extragenic spacer length and not its sequence because if the sequence was inserted into the expression cassette so that it became part of the expressed 3'UTR, silencing was not observed (Discussion, first paragraph, page 2117).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to try plasmid backbones with 1 kb or less taught by Lu et al. as the vector in the method taught by Slilaty to study transgene expression and avoid gene silencing in cells. One of ordinary skill in the art would have been motivated to produce a plasmid vector containing an expression cassette sequence encoding the lacZ α-peptide operably linked to the lac promoter that is less than 1.5 kb in size. Doing so would improve the transgene expression through reduced silencing, as recognized by Lu. Further, as the DNA sequence encoding lacZ taught by Slilaty appears to be less than 0.5 kb of the vector taught by Slilaty, there is a reasonable expectation that the extragenic sequence could be reduced to less than 1 kb and achieve a total vector size of less than 1.5 kb.
Therefore the invention as a whole would have been prima facie obvious to one ordinary skill in the art before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains.
Response to Arguments
Applicant's arguments filed 6/24/25 have been fully considered but they are not persuasive because the only arguments against this additional reference is directed at Slilaty. The argument is not found persuasive for the reasons set forth above in response to Slilaty and incorporated herein.
Allowable Subject Matter
Claim 17 is objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims. The prior art does not teach0 or suggest a vector comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9-13 and 17-18.
Conclusion
See attached PTO-326 for disposition of claims.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. Lee et al. (US 20050019857) teach growing an expression vector comprising an OmpF promoter operably linked to a nucleotide sequence encoding a fusion protein comprising a protein of interest for extracellular production of the protein of interest. The vector was grown in E. coli cells (XL1-blue) having a laczM15 (paragraphs 21-26). ‘857 does not appear to teach or suggest the nucleotide sequence encoding an amino acid sequence having at least 95% to SEQ ID NO: 1 in the vector.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Brian Whiteman whose telephone number is (571)272-0764. The examiner can normally be reached on Monday thru Friday; 6:00 AM to 3:00PM.
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/BRIAN WHITEMAN/ Primary Examiner, Art Unit 1636
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