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 .
Priority
The instant application filed on 6/20/2022 claims benefit to Chinese application CN202210331773.2 filed on 3/31/2022. Thus, the effective filing date for claims 1-8 is 3/21/2022.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 6/20/2022 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements have been considered by the examiner. The IDS submitted on 3/13/2023 was a duplicate of the IDS filed on 6/20/2022 and therefore was not considered.
Status of Claims
Claims 1-8 are pending.
Claims 1-8 are rejected.
Drawings
The Drawings filed on 6/20/2022 and 10/13/2022 are not in compliance due Figures 1, 4, 5, 6, 7, 8,9 containing sequences with a length greater than 10, without the accompanied SEQ ID either in the drawing itself or in the brief description of the drawing.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-8 are rejected under 35 U.S.C. 101 because the claimed invention is directed to an abstract idea without significantly more. The claims recite: (a) mathematical concepts, (e.g., mathematical relationships, formulas or equations, mathematical calculations); and (b) mental processes, i.e., concepts performed in the human mind, (e.g., observation, evaluation, judgement, opinion).
Subject matter eligibility evaluation in accordance with MPEP 2106:
Eligibility Step 1: Claims 1-8 are directed to method for screening split sites.
[Step 1: YES]
Eligibility Step 2A: First it is determined in Prong One whether a claim recites a judicial exception, and if so, then it is determined in Prong Two whether the recited judicial exception is integrated into a practical application of that exception.
Eligibility Step 2A Prong One: In determining whether a claim is directed to a judicial exception, examination is performed that analyzes whether the claim recites a judicial exception, i.e., whether a law of nature, natural phenomenon, or abstract idea is set forth or described in the claim.
Independent
Independent claim 1 recites the following steps which fall within the mental processes and/or mathematical concepts groupings of abstract ideas:
predicting an amino acid sequence formed by connecting adjacent peptide fragments after an intein is embedded into each two adjacent amino acid residues in an initial amino acid sequence and then excised through a self-splicing reaction to construct the protein database (mental processes and/or mathematical concepts)
comparing the peptide fragment with the protein database when the peptide fragment is detected as containing the labeled amino acid sequence to confirm the split site (mental processes and/or mathematical concepts)
Dependent claim 2 recites the following steps which fall within the mental processes and/or mathematical concepts groupings of abstract ideas:
searching a target intein amino acid sequence in the new amino acid sequence (mental processes and/or mathematical concepts)
predicting each possible site of the first gene segment and the second gene segment into which the inserted intein sequence segment is inserted (mental processes and/or mathematical concepts)
Dependent claim 6 recites the following steps which fall within the mental processes and/or mathematical concepts groupings of abstract ideas:
searching a target intein amino acid sequence in the new amino acid sequence (mental processes and/or mathematical concepts)
predicting each possible site of the first gene segment and the second gene segment into which the inserted intein sequence segment is inserted (mental processes and/or mathematical concepts)
The abstract ideas recited in the claims are evaluated under the broadest reasonable interpretation (BRI) of the claim limitations when read in light of and consistent with the specification. As noted in the foregoing section, the claims are determined to contain limitations that can practically be performed in the human mind with the aid of a pencil and paper, and therefore recite judicial exceptions from the mental process grouping of abstract ideas. Additionally, the recited limitations that are identified as judicial exceptions from the mathematical concepts grouping of abstract ideas are abstract ideas irrespective of whether or not the limitations are practical to perform in the human mind.
Therefore, claims 1-8 recite an abstract idea as the dependent claims will inherit the abstract ideas from the independent claims.
[Step 2A Prong One: YES]
Eligibility Step 2A Prong Two: In determining whether a claim is directed to a judicial exception, further
examination is performed that analyzes if the claim recites additional elements that when examined as a
whole integrates the judicial exception(s) into a practical application (MPEP 2106.04(d)). A claim that
integrates a judicial exception into a practical application will apply, rely on, or use the judicial exception
in a manner that imposes a meaningful limit on the judicial exception. The claimed additional elements
are analyzed to determine if the abstract idea is integrated into a practical application (MPEP
2106.04(d)(I); MPEP 2106.05(a-h)). If the claim contains no additional elements beyond the abstract
idea, the claim fails to integrate the abstract idea into a practical application (MPEP 2106.04(d)(III)).
The judicial exceptions identified in Eligibility Step 2A Prong One are not integrated into a practical application because of the reasons noted below.
The additional element in independent claim 1 includes:
Writing a program by using a computer language
inserting an intein sequence into a gene segment through a molecular clone experimental method
then translating to obtain a peptide fragment
detecting whether that the peptide fragment contains a labeled amino acid sequence by mass spectrometry
The additional element in dependent claim 2 includes:
fusing a first gene segment, an inserted intein sequence segment, and a second gene segment in a sequential order to obtain a new deoxyribonucleic acid (DNA) sequence
translating the new DNA sequence into a new amino acid sequence
deleting the target intein amino acid sequence in the new amino acid sequence to thereby obtain an output amino acid sequence
repeating the steps S11 to S13 to obtain all the output amino acid sequences to construct the protein data database
The additional element in dependent claim 3 includes:
wherein in the step S1, at least one base is inserted into the inserted intein sequence segment.
The additional element in dependent claim 4 includes:
wherein the at least one base is one base
The additional element in dependent claim 5 includes:
in screening split sites of at least one of Escherichia coli (E.coli) antigen protein Im7-6 and Cas9 protein, wherein Im7-6 refers to immunity protein 7-6, and Cas9 refers to clustered regularly interspaced short palindromic repeats associated protein 9.
The additional element in dependent claim 6 includes:
fusing a first gene segment, an inserted intein sequence segment, and a second gene segment in a sequential order to obtain a new deoxyribonucleic acid (DNA) sequence
translating the new DNA sequence into a new amino acid sequence
deleting the target intein amino acid sequence in the new amino acid sequence to thereby obtain an output amino acid sequence
repeating the steps S11 to S13 to obtain all the output amino acid sequences to construct the protein data database
The additional element in dependent claim 7 includes:
at least one base is inserted into the inserted intein sequence segment
The additional element in dependent claim 8 includes:
wherein the at least one base is one base
The additional elements of writing a program by using a computer language (claim 1) merely invokes a computer as a tool and does not improve the technology of a generic computer (see MPEP 2106.05(a). The additional elements of inserting an intein sequence into a gene segment through a molecular clone experimental method (claim 1) then translating to obtain a peptide fragment (claim 1), detecting whether that the peptide fragment contains a labeled amino acid sequence by mass spectrometry (claim 1), fusing a first gene segment, an inserted intein sequence segment, and a second gene segment in a sequential order to obtain a new deoxyribonucleic acid (DNA) sequence (claim 2) translating the new DNA sequence into a new amino acid sequence (claim 2) deleting the target intein amino acid sequence in the new amino acid sequence to thereby obtain an output amino acid sequence (claim 2) repeating the steps S11 to S13 to obtain all the output amino acid sequences to construct the protein data database (claim 2), wherein in the step S1, at least one base is inserted into the inserted intein sequence segment. (claim 3), wherein the at least one base is one base (claim 4), in screening split sites of at least one of Escherichia coli (E.coli) antigen protein Im7-6 and Cas9 protein, wherein Im7-6 refers to immunity protein 7-6, and Cas9 refers to clustered regularly interspaced short palindromic repeats associated protein 9. (claim 5), fusing a first gene segment, an inserted intein sequence segment, and a second gene segment in a sequential order to obtain a new deoxyribonucleic acid (DNA) sequence (claim 6), translating the new DNA sequence into a new amino acid sequence (claim 6), searching a target intein amino acid sequence in the new amino acid sequence, and deleting the target intein amino acid sequence in the new amino acid sequence to thereby obtain an output amino acid sequence (claim 6), repeating the steps S11 to S13 to obtain all the output amino acid sequences to construct the protein data database (claim 6), at least one base is inserted into the inserted intein sequence segment (claim 7), wherein the at least one base is one base (claim 8) are all insignificant extra-solution activity that are part of the data gathering process used in the recited judicial exceptions (see MPEP 2106.05(g)). As all these steps are for the purpose of gathering data for a protein database or information to compare to the protein database.
Claims 1-8 do not recite any elements in addition to the judicial exception, and thus are part of the judicial exception.
Thus, the additionally recited elements merely invoke a computer as a tool, and/or amount to insignificant extra-solution data gathering activity, and as such, when all limitations in claims 1-8 have been considered as a whole, the claims are deemed to not recite any additional elements that would integrate a judicial exception into a practical application, and therefore claims 1-8 are directed to an abstract idea (MPEP 2106.04(d)).
[Step 2A Prong Two: NO]
Eligibility Step 2B: Because the claims recite an abstract idea, and do not integrate that abstract idea into a practical application, the claims are probed for a specific inventive concept. The judicial exception alone cannot provide that inventive concept or practical application (MPEP 2106.05). Identifying whether the additional elements beyond the abstract idea amount to such an inventive concept requires considering the additional elements individually and in combination to determine if they amount to significantly more than the judicial exception (MPEP 2106.05A i-vi).
The claims do not include any additional elements that are sufficient to amount to significantly more than the judicial exception(s) because of the reasons noted below.
The additional elements recited in claims 1-8 are identified above, and carried over from Step 2A: Prong Two along with their conclusions for analysis at Step 2B. Any additional element or combination of elements that was considered to be insignificant extra-solution activity at Step 2A: Prong Two was re-evaluated at Step 2B, because if such re-evaluation finds that the element is unconventional or otherwise more than what is well-understood, routine, conventional activity in the field, this finding may indicate that the additional element is no longer considered to be insignificant; and all additional elements and combination of elements were evaluated to determine whether any additional elements or combination of elements are other than what is well-understood, routine, conventional activity in the field, or simply append well-understood, routine, conventional activities previously known to the industry, specified at a high level of generality, to the judicial exception, per MPEP 2106.05(d).
The additional elements of writing a program by using a computer language (claim 1) merely invokes a computer as a tool and does not improve the technology of a generic computer (see MPEP 2106.05(a). The additional elements of inserting an intein sequence into a gene segment through a molecular clone experimental method (claim 1) then translating to obtain a peptide fragment (claim 1), detecting whether that the peptide fragment contains a labeled amino acid sequence by mass spectrometry (claim 1), fusing a first gene segment, an inserted intein sequence segment, and a second gene segment in a sequential order to obtain a new deoxyribonucleic acid (DNA) sequence (claim 2) translating the new DNA sequence into a new amino acid sequence (claim 2) deleting the target intein amino acid sequence in the new amino acid sequence to thereby obtain an output amino acid sequence (claim 2) repeating the steps S11 to S13 to obtain all the output amino acid sequences to construct the protein data database (claim 2), wherein in the step S1, at least one base is inserted into the inserted intein sequence segment. (claim 3), wherein the at least one base is one base (claim 4), in screening split sites of at least one of Escherichia coli (E.coli) antigen protein Im7-6 and Cas9 protein, wherein Im7-6 refers to immunity protein 7-6, and Cas9 refers to clustered regularly interspaced short palindromic repeats associated protein 9. (claim 5), fusing a first gene segment, an inserted intein sequence segment, and a second gene segment in a sequential order to obtain a new deoxyribonucleic acid (DNA) sequence (claim 6), translating the new DNA sequence into a new amino acid sequence (claim 6), searching a target intein amino acid sequence in the new amino acid sequence, and deleting the target intein amino acid sequence in the new amino acid sequence to thereby obtain an output amino acid sequence (claim 6), repeating the steps S11 to S13 to obtain all the output amino acid sequences to construct the protein data database (claim 6), at least one base is inserted into the inserted intein sequence segment (claim 7), wherein the at least one base is one base (claim 8) are conventional and are insignificant extra-solution activity that are part of the data gathering process used in the recited judicial exceptions (see MPEP 2106.05(g)). Evidence for conventionality is shown by by Lee et al. (Lee, Y.-T.; et al. Circular Permutation Prediction Reveals a Viable Backbone Disconnection for Split Proteins: An Approach in Identifying a New Functional Split Intein. PLoS ONE 2012, 7 (8), e43820.) which is a computer program for identifying functional split inteins, Ho et al. (Ho et al. Systematic Approach to Inserting Split Inteins for Boolean Logic Gate Engineering and Basal Activity Reduction. Nature Communications 2021, 12 (1)) a method for inserting split inteins and Lockless et al. (Lockless, S. W.; Muir, T. W. Traceless Protein Splicing Utilizing Evolved Split Inteins. Proceedings of the National Academy of Sciences 2009, 106 (27), 10999–11004) a method of protein splicing utilizing evolved split inteins and Truong et al. (Truong, D.-J. J.; et al. Development of an Intein-Mediated Split–Cas9 System for Gene Therapy. Nucleic Acids Research 2015, 43 (13), 6450–6458.) which is a method to develop an intein-mediated split–Cas9 system for gene therapy all use the same basic laboratory techniques described above and thus are conventional.
Claims 1-8 do not recite any elements in addition to the judicial exception.
Therefore, when taken alone, all additional elements in claims 1-8 do not amount to significantly more than the above-identified judicial exception(s). Even when evaluated as a combination, the additional elements fail to transform the exception(s) into a patent-eligible application of that exception. Thus, claims 1-8 are deemed to not contribute an inventive concept, i.e., amount to significantly more than the judicial exception(s) (MPEP 2106.05(II)).
[Step 2B: NO]
Claim Rejections - 35 USC § 103
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.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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 1-4 are rejected under 35 U.S.C. 103 as being unpatentable by Lee et al. (Lee, Y.-T.; et al. Circular Permutation Prediction Reveals a Viable Backbone Disconnection for Split Proteins: An Approach in Identifying a New Functional Split Intein. PLoS ONE 2012, 7 (8), e43820.) in view of Ho et al. (Ho et al. Systematic Approach to Inserting Split Inteins for Boolean Logic Gate Engineering and Basal Activity Reduction. Nature Communications 2021, 12 (1)) in view of Lockless et al. (Lockless, S. W.; Muir, T. W. Traceless Protein Splicing Utilizing Evolved Split Inteins. Proceedings of the National Academy of Sciences 2009, 106 (27), 10999–11004)
Regarding the limitations of independent claim 1,
A method for screening a split site, comprising: step Si, establishing a protein database, which comprises: writing a program by using a computer language
Lee at al. teaches that Circular permutation (CP) is an emerging method designed to introduce changes into protein sequences and thus protein structure (Introduction 1st paragraph, pg. 1, col. 1) developed an efficient program, CPSARST (Circular Permutation Search Aided by Ramachandran Sequential Transformation), to search target protein for CPs in databases. (Introduction 2nd paragraph, pg. 1, col. 2)
predicting an amino acid sequence formed by connecting adjacent peptide fragments after an intein is embedded into each two adjacent amino acid residues in an initial amino acid sequence and then excised through a self-splicing reaction
Lee at al. teaches that Circular permutation (CP) is an emerging method designed to introduce changes into protein sequences and thus protein structure (Introduction 1st paragraph, pg. 1, col. 1) and CPred successfully predicted the folding of both naturally occurring and artificially designed CPs (Introduction 2nd paragraph, pg. 1, col. 2) and they showed that a valid CP site can be used as a split site ((Introduction 3rd paragraph, pg. 1, col. 2) and they analyzed three commonly used inteins using CPred and correlated our results with previously reported split sites that support efficient PTS reactions CPred outputs a value between 0 and 1 for each residue, with scores closer to 1 corresponding to an increased likelihood for CP. We noticed that almost all of the functional split sites were at or near sites with local CPred score maximums. (Assessment of the Intein CP Site Prediction, paragraph 1, pg. 2, col. 1). This teaches a computational method that identifies specific positions between amino acids where splitting can occur which is equivalent to predicting where an intein can be embedded into each two adjacent amino acid residues.
Lee et al. does not explicitly teach
to construct the protein database (claim 1)
step S2, performing an experiment, which comprises: inserting an intein sequence into a gene segment through a molecular clone experimental method (claim 1)
then translating to obtain a peptide fragment (claim 1)
comparing the peptide fragment with the protein database when the peptide fragment is detected as containing the labeled amino acid sequence to confirm the split site (claim 1)
detecting whether that the peptide fragment contains a labeled amino acid sequence by mass spectrometry (claim 1)
wherein in the step Si, the establishing a protein database specifically comprises: step S11, fusing a first gene segment, an inserted intein sequence segment, and a second gene segment in a sequential order to obtain a new deoxyribonucleic acid (DNA) sequence; step S12, translating the new DNA sequence into a new amino acid sequence; step S13, searching a target intein amino acid sequence in the new amino acid sequence, and deleting the target intein amino acid sequence in the new amino acid sequence to thereby obtain an output amino acid sequence; and step S14, predicting each possible site of the first gene segment and the second gene segment into which the inserted intein sequence segment is inserted, and repeating the steps S11 to S13 to obtain all the output amino acid sequences to construct the protein data database (claim 2)
wherein in the step S1, at least one base is inserted into the inserted intein sequence segment. (claim 3)
wherein the at least one base is one base (claim 4)
Regarding the limitations of independent claim 1,
to construct the protein database
Ho et al. teaches that pooling the results yielded an intein-bisection map. A total of 15 split sites were identified. All split sites clustered into four seams (pg. 3, col. 1, 2nd paragraph) and Three split seams and 32 split sites were found for TetR; 4 seams and 13 sites for SrpR; 3 seams and 17 sites for ECF20 (pg 4, col. 1, Applying IBM to engineer AND and NAND logic gates, 1st paragraph). This is a collection or database of split site information.
step S2, performing an experiment, which comprises: inserting an intein sequence into a gene segment through a molecular clone experimental method
Ho et al. teaches the method that starts with an in vitro transposition reaction that randomly inserts a BbsI and SapI-flanked transposon into a staging vector, which hosts a slightly trimmed, BsaI-flanked coding DNA sequence (CDS) of interest. This is followed by size selection of the insertion library such that only CDS fragments with insertions will be isolated and ligated into a vector for protein expression. A Golden Gate reaction was then used to irreversibly substitute the transposon with a DNA fragment. The fragment carries a selection marker, a split intein, and transcription and translation initiation elements for carboxyl-lobe (Clobe) expression. In-frame insertions in the right orientation will thus split a CDS into two with the amino-lobes (N-lobes) and Clobes of the split intein as fusion partners, under separate control of two inducible promoters. The final library is then screened for individual clones that display functional activities only when the chemical inducers for both promoters are present. The clones are then sequenced at the fusion joints to reveal the split sites. (pg. 2, Designing the IBM workflow for split site screening, paragraph 1, col. 2). A person having ordinary skill in the art would understand this describes a molecular cloning method that inserts an intein sequence.
then translating to obtain a peptide fragment
Ho et al. teaches in-frame insertions in the right orientation will thus split a CDS into two with the amino-lobes (N-lobes) and Clobes of the split intein as fusion partners, under separate control of two inducible promoters (pg. 2, Designing the IBM workflow for split site screening, paragraph 1, col. 2). This approach expresses proteins/ peptides.
comparing the peptide fragment with the protein database when the peptide fragment is detected as containing the labeled amino acid sequence to confirm the split site
Ho et al. teaches cells with fluorescence above autofluorescence were sorted by fluorescence-activated cell sorting (FACS), plated and isolated as single colonies. Individual strains were then assayed for responses in the absence or presence of the two inducers, and those that showed AND logic behavior were subsequently sequenced to identify the split sites. Pooling the results yielded an intein-bisection map. (pg. 3, col. 1, paragraph 2) Sequencing results are compared against the expected intein bisection map to identify which split site is present. The fluorescence indicates successful peptide fragment formation. The split site was confirmed with sequencing. The sequencing revealed which amino acid sequence is present which is compared to the possible split sites in order to confirm which split site was successfully used.
Regarding the limitations of dependent claim 2,
wherein in the step Si, the establishing a protein database specifically comprises: step S11, fusing a first gene segment, an inserted intein sequence segment, and a second gene segment in a sequential order to obtain a new deoxyribonucleic acid (DNA) sequence
Ho et al. teaches the method that starts with an in vitro transposition reaction that randomly inserts a BbsI and SapI-flanked transposon into a staging vector, which hosts a slightly trimmed, BsaI-flanked coding DNA sequence (CDS) of interest. This is followed by size selection of the insertion library such that only CDS fragments with insertions will be isolated and ligated into a vector for protein expression. A Golden Gate reaction was then used to irreversibly substitute the transposon with a DNA fragment. The fragment carries a selection marker, a split intein, and transcription and translation initiation elements for carboxyl-lobe (Clobe) expression. In-frame insertions in the right orientation will thus split a CDS into two with the amino-lobes (N-lobes) and Clobes of the split intein as fusion partners, under separate control of two inducible promoters. The final library is then screened for individual clones that display functional activities only when the chemical inducers for both promoters are present. The clones are then sequenced at the fusion joints to reveal the split sites. (pg. 2, Designing the IBM workflow for split site screening, paragraph 1, col. 2). A person having ordinary skill in the art would understand this creates a fusion of gene segment + intein + gene segment.
step S12, translating the new DNA sequence into a new amino acid sequence
Ho et al. teaches the method that starts with an in vitro transposition reaction that randomly inserts a BbsI and SapI-flanked transposon into a staging vector, which hosts a slightly trimmed, BsaI-flanked coding DNA sequence (CDS) of interest. This is followed by size selection of the insertion library such that only CDS fragments with insertions will be isolated and ligated into a vector for protein expression. A Golden Gate reaction was then used to irreversibly substitute the transposon with a DNA fragment. The fragment carries a selection marker, a split intein, and transcription and translation initiation elements for carboxyl-lobe (Clobe) expression. In-frame insertions in the right orientation will thus split a CDS into two with the amino-lobes (N-lobes) and Clobes of the split intein as fusion partners, under separate control of two inducible promoters. The final library is then screened for individual clones that display functional activities only when the chemical inducers for both promoters are present. The clones are then sequenced at the fusion joints to reveal the split sites. (pg. 2, Designing the IBM workflow for split site screening, paragraph 1, col. 2). Fluorescence was used to confirm. (pg. 3, col. 1, paragraph 2) Translation will produce the protein.
step S13, searching a target intein amino acid sequence in the new amino acid sequence, and deleting the target intein amino acid sequence in the new amino acid sequence to thereby obtain an output amino acid sequence
Ho et al. teaches protein splicing took place at all split sites as evidenced by a Western blot experiment on whole-cell lysates, and we observed that all C-lobes were consumed. This proved that our IBM workflow can locate intein insertion sites that support efficient splicing. (pg. 3 col 2. Paragraph 1) Successful splicing of the M86 intein would leave behind a highly predictable fourresidue peptide linker at the split site of the original protein (Supplementary Fig. 3). Our method emphasizes the use of an intein in bisection mapping—hence the name intein-assisted bisection mapping (IBM). (pg. 2, col. 2, Designing the IBM workflow for split site screening, paragraph 2)
step S14, predicting each possible site of the first gene segment and the second gene segment into which the inserted intein sequence segment is inserted and repeating the steps S11 to S13 to obtain all the output amino acid sequences to construct the protein data database
Ho et al. teaches that their final libraries prior to screening, sequenced by Next Generation Sequencing (NGS), we obtained at least 87% coverage on possible amino acid split/insertion positions which sufficiently explored the sequence space. (pg. 9, col. 1, paragraph, 3) These are predicted split sites. A total of 15 split sites were identified (pg. 3, col. 1, paragraph 2) and pooling the results yielded an intein-bisection map. A total of 15 split sites were identified. All split sites clustered into four seams (pg. 3, col. 1, 2nd paragraph) and Three split seams and 32 split sites were found for TetR; 4 seams and 13 sites for SrpR; 3 seams and 17 sites for ECF20 (pg 4, col. 1, Applying IBM to engineer AND and NAND logic gates, 1st paragraph). This is a collection or database of split site information.
Regarding the limitations of dependent claim 3,
wherein in the step S1, at least one base is inserted into the inserted intein sequence segment.
Ho et al. teaches that the fragment carries a selection marker, a split intein, and transcription and translation initiation elements for carboxyl-lobe (Clobe) expression. (pg. 2, col. 2, Designing the IBM workflow for split site screening, paragraph 1). The transcription and translation would include regulatory sequences, start codons, and potentially additional bases. Therefore, they are bases inserted into the intein sequence segment.
Regarding the limitations of dependent claim 4,
wherein the at least one base is one base.
Ho et al. teaches that the fragment carries a selection marker, a split intein, and transcription and translation initiation elements for carboxyl-lobe (Clobe) expression. (pg. 2, col. 2, Designing the IBM workflow for split site screening, paragraph 1). The transcription and translation would include regulatory sequences, start codons, and potentially additional bases. Therefore, they are bases inserted into the intein sequence segment. A single base is obvious because it is optimization of a known range and Ho et al. provides a general method for inserting an intein of variable sizes.
Regarding the limitations of independent claim 1,
detecting whether that the peptide fragment contains a labeled amino acid sequence by mass spectrometry
Lockless et al. teaches the Npu* reaction led to the appearance of both the spliced product and the 16-kDa band, which was confirmed as the branched intermediate by mass spectrometry (pg. 11000, col. 2, first paragraph)
A person having ordinary skill in the art would be motivated to combine the prior art of methods of Lee et al., Ho et al., and Lockless et al. because all are involved with identifying split sites for inteins. These references represent different parts of a standard scientific toolkit, computational prediction, experimental screening, and analytical validation. Additionally, when combined each method is performing the same function as it did separately and thus will yield the same predictable results because different methods do not modify the steps when combined.
Claims 5-8 are rejected under 35 U.S.C. 103 as being unpatentable over Lee et al. in view of Ho et al. in view of Lockless et al. as applied to claims 1-4 above, and further in view of Truong et al. (Truong, D.-J. J.; et al. Development of an Intein-Mediated Split–Cas9 System for Gene Therapy. Nucleic Acids Research 2015, 43 (13), 6450–6458.)
As applied to claims 1-4 (detailed above), Lee et al. in view of Ho et al. in view of Lockless et al. teaches a method for screening split sites.
Regarding the limitations of dependent claim 6,
wherein in the step Si, the establishing a protein database specifically comprises: step S11, fusing a first gene segment, an inserted intein sequence segment, and a second gene segment in a sequential order to obtain a new deoxyribonucleic acid (DNA) sequence; step S12, translating the new DNA sequence into a new amino acid sequence; step S13, searching a target intein amino acid sequence in the new amino acid sequence, and deleting the target intein amino acid sequence in the new amino acid sequence to thereby obtain an output amino acid sequence; and step S14, predicting each possible site of the first gene segment and the second gene segment into which the inserted intein sequence segment is inserted, and repeating the steps S11 to S13 to obtain all the output amino acid sequences to construct the protein data database.
This is equivalent to claim 2 except applied to a specific protein, please see Lee et al. in view of Ho et al. in view of Lockless et al. as applied to claim 2 above.
Regarding the limitations of dependent claim 7,
wherein in the step 511, at least one base is inserted into the inserted intein sequence segment.
This is equivalent to claim 3 except applied to a specific protein, please see Lee et al. in view of Ho et al. in view of Lockless et al. as applied to claim 3 above.
Regarding the limitations of dependent claim 8,
wherein the at least one base is one base.
This is equivalent to claim 4 except applied to a specific protein, please see Lee et al. in view of Ho et al. in view of Lockless et al. as applied to claim 4 above.
Lee et al. in view of Ho et al. in view of Lockless et al. does not explicitly teach:
the method according to claim 1 in screening split sites of at least one of Escherichia coli (E.coli) antigen protein Im7-6 and Cas9 protein, wherein Im7-6 refers to immunity protein 7-6, and Cas9 refers to clustered regularly interspaced short palindromic repeats associated protein 9 (Claim 5).
Regarding the limitations of dependent claim 5,
the method according to claim 1 in screening split sites of at least one of Escherichia coli (E.coli) antigen protein Im7-6 and Cas9 protein, wherein Im7-6 refers to immunity protein 7-6, and Cas9 refers to clustered regularly interspaced short palindromic repeats associated protein 9.
Truong et al. teaches the use a naturally occurring phenomenon by exchanging the extein regions with the respective halves of SpCas9. The split-sites of SpCas9 were carefully chosen between Glu573 and Cys574 for the first version (v1) or between Lys637 and Thr638 for the second version (v2), since the N-terminal amino acid of the C-Cas9 in the C-Intein C-Cas9 fusion should be Cys, Ser or Thr to ensure high splicing efficiency. (pg. 6452 col. 1-2, Design of Intein-mediated split SpCas9, paragraph 1) This work incorporates the use of Cas9 protein.
A person having ordinary skill in the art would be motivated to use the method of method for screening split sites of Lee et al., Ho et al., and Lockless et al. with the knowledge of screening split sites of Escherichia coli Cas9 protein. It would be obvious for a person having ordinary skill in the art to use a known method with a target of Cas9 protein which Truong et al. used for intein-mediated split–Cas9 for gene therapy. Additionally, when combined each method is performing the same function as it did separately and thus will yield the same predictable results.
Conclusion
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/C.H.B./Examiner, Art Unit 1687
/Karlheinz R. Skowronek/Supervisory Patent Examiner, Art Unit 1687