CTNF 17/641,835 CTNF 101607 Notice of Pre-AIA or AIA Status 07-03-aia AIA 15-10-aia 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 the Claims Claims 1-20 are currently pending and under exam herein. Claims 1-20 are rejected. Priority The instant application is a 371 of PCT/EP2020/075535 which claims priority to foreign application DE102019124459.5 filed on 9/11/2019. Thus, the effective filing date of the instant application is 9/11/2019. Drawings The Drawings filed on 9/19/2022 were considered. Information Disclosure Statement The information disclosure statement (IDS) submitted on 3/22/2022 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement has been considered by the examiner. Claim Rejections - 35 USC § 101 07-04-01 AIA 07-04 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 1-20 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 1-16, 18-20 are directed to a to a method for screening pore-forming membrane proteins, membrane transporters and molecular switches. Claim 17 is directed towards a system to carry out a method for screening pore-forming membrane proteins, membrane transporters and molecular switches. [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 claim 1 recites the following steps which fall within the mental processes and/or mathematical concepts groupings of abstract ideas: calculating one or more comparative parameters from the signal strengths detected in steps b) and d), wherein step b) is carried out before step c) and wherein step d) is carried out after step c), (mathematics) wherein steps a) to e) are carried out for at least two membrane proteins having different amino acid sequences and/or for at least two sensor proteins having different amino acid sequences. (mathematics, this is just limiting what mathematics done now) Independent claim 14 recites the following steps which fall within the mental processes and/or mathematical concepts groupings of abstract ideas: wherein step d) is carried out at least five times, and the calculation of the comparative parameter from the signal strengths according to step e) includes empirically fitting the data obtained on the signal strengths with a sigmoid function, wherein the comparative parameter calculated from the signal strengths is the half-maximum time c and/or the maximum slope d. (mathematics) Independent claim 15 recites the following steps which fall within the mental processes and/or mathematical concepts groupings of abstract ideas: wherein the data obtained on the signal strengths are fitted empirically with the following equation 1 PNG media_image1.png 127 517 media_image1.png Greyscale (mathematics) 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 1-20 recites 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: A screening method comprising the steps of: a) providing a cell and a medium surrounding the cell, - wherein the cell has a cell membrane impermeable to a reporter substance, - wherein the quotient of the concentration of the reporter substance in the interior of the cell and the concentration of the reporter substance in the medium surrounding the cell is at least 2 or at most 0.5; detecting a signal that is dependent on the concentration of the reporter substance in the cell and that is generated with the aid of a sensor protein expressed in the cell; inducing the expression of a membrane protein that increases the permeability of the cell membrane to the reporter substance; detecting the signal that is dependent on the concentration of the reporter substance in the cell and that is generated with the aid of the sensor protein expressed in the cell; The additional element in independent claim 2 includes: comprising the further step of: f) determining the DNA sequence encoding the membrane protein and/or the DNA sequence encoding the sensor protein. The additional element in independent claim 3 includes: wherein the cell is a microorganism. The additional element in independent claim 4 includes: wherein the cell is Escherichia coli, and the cell the cell membrane is the inner membrane of E. coli. The additional element in independent claim 5 includes: wherein steps a) to f) are carried out for at least 5 membrane proteins with different amino acid sequences. The additional element in independent claim 6 includes: wherein steps a) to f) are carried out for at least 5 sensor proteins with different amino acid sequences. The additional element in independent claim 7 includes: wherein the signal is selected from one or more of a fluorescence signal, a bioluminescence signal, and an absorption signal. The additional element in independent claim 8 includes: wherein the sensor protein is selected from the group of Ca 2+ -specific protein sensors and L-Glu-specific protein sensors. The additional element in independent claim 9 includes: wherein the reporter substance is selected from the group consisting of cations and amino acids. The additional element in independent claim 10 includes: wherein the membrane protein is selected from the group consisting of pore-forming membrane proteins and membrane transporters. The additional element in independent claim 11 includes: wherein the membrane protein is selected from the group consisting of holins, pinholins, and ion channels. The additional element in independent claim 12 includes: wherein the membrane protein is selected from the group consisting of S 21 -68 (SEQ ID NO:1), S 21 -71 (SEQ ID NO:2), S 21 -71 M4A (SEQ ID NO:3), SGS ΔTMD1S 21 68 (SEQ ID NO:4), TVMV ΔTMD1-S 21 68 (SEQ ID NO:5), S 105 (SEQ ID NO:6), S 107 (SEQ ID NO:7), S 107 -M3A (SEQ ID NO:8), T4 pinholin (SEQ ID NO:9), T4 ΔC-Tail (SEQ ID NO:10), ΔN-Tail T4 ΔC-Tail (SEQ ID NO:11), K CV NTS (SEQ ID NO: 2), K CV NTS G77S (SEQ ID NO:13), K CV PBCV1 (SEQ ID NO:14), HokB (SEQ ID NO:15), TisB (SEQ ID NO:16), αHLA (SEQ ID NO:17), cWZA (SEQ ID NO:18), BM2 (SEQ ID NO:19), HCV TME1 (SEQ ID NQ:20), HCV TME2 (SEQ ID NO:21) and variants thereof having sequence identity to at least one of the sequences of SEQ ID NO: 1-21 of at least 90%. The additional element in independent claim 13 includes: wherein step d) is carried out at most 120 minutes after step c). The additional element in independent claim 16 includes: screening method according to claim 1, wherein the cell is a prokaryotic microorganism. The additional element in independent claim 17 includes: kit for carrying out the screening method according to claim 1, the kit comprising: i. DNA encoding at least two membrane proteins and DNA encoding a sensor protein, or ii. DNA encoding at least two sensor proteins and DNA encoding a membrane protein. The additional element in independent claim 18 includes: screening method according to claim 2, wherein steps a) to f) are carried out for at least 20 membrane proteins with different amino acid sequences. The additional element in independent claim 19 includes: wherein steps a) to f) are carried out for at least 50 membrane proteins with different amino acid sequences. The additional element in independent claim 20 includes: wherein steps a) to f) are carried out for at least 10 2 to 10 8 membrane proteins with different amino acid sequences The additional elements of claims 1-13 and 18-20, stated above , are insignificant extra-solution activity that are part of the data gathering process used in the recited judicial exceptions or simply limit the type of data being gathered (see MPEP 2106.05(g)). Claim 17, stated above , are directed to a kit for the purpose of data gathering process used in the recited judicial exceptions to carry out insignificant extra-solution activity to carry out an abstract idea (see MPEP 2106.05(g)). Claims 14-15 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 1-20 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 1-20 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 1-13 and 16-20 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 a) providing a cell and a medium surrounding the cell, - wherein the cell has a cell membrane impermeable to a reporter substance, - wherein the quotient of the concentration of the reporter substance in the interior of the cell and the concentration of the reporter substance in the medium surrounding the cell is at least 2 or at most 0.5 (Claim 1), detecting a signal that is dependent on the concentration of the reporter substance in the cell and that is generated with the aid of a sensor protein expressed in the cell; (Claim 1), inducing the expression of a membrane protein that increases the permeability of the cell membrane to the reporter substance; (Claim 1), detecting the signal that is dependent on the concentration of the reporter substance in the cell and that is generated with the aid of the sensor protein expressed in the cell; (Claim 1), wherein the cell is a microorganism (Claim 3), wherein the cell is Escherichia coli, and the cell membrane is the inner membrane of E. coli. (Claim 4), wherein the signal is selected from one or more of a fluorescence signal, a bioluminescence signal, and an absorption signal (Claim 7), wherein the sensor protein is selected from the group of Ca 2+ -specific protein sensors and L-Glu-specific protein sensors. (Claim 8), wherein the reporter substance is selected from the group consisting of cations and amino acids (Claim 9), wherein the membrane protein is selected from the group consisting of pore-forming membrane proteins and membrane transporters. (Claim 10), wherein the membrane protein is selected from the group consisting of holins, pinholins, and ion channels. (Claim 11), wherein the membrane protein is selected from the group consisting of S 21 -68 (SEQ ID NO:1), S 21 -71 (SEQ ID NO:2), S 21 -71 M4A (SEQ ID NO:3), SGS ΔTMD1S 21 68 (SEQ ID NO:4), TVMV ΔTMD1-S 21 68 (SEQ ID NO:5), S 105 (SEQ ID NO:6), S 107 (SEQ ID NO:7), S 107 -M3A (SEQ ID NO:8), T4 pinholin (SEQ ID NO:9), T4 ΔC-Tail (SEQ ID NO:10), ΔN-Tail T4 ΔC-Tail (SEQ ID NO:11), K CV NTS (SEQ ID NO: 2), K CV NTS G77S (SEQ ID NO:13), K CV PBCV1 (SEQ ID NO:14), HokB (SEQ ID NO:15), TisB (SEQ ID NO:16), αHLA (SEQ ID NO:17), cWZA (SEQ ID NO:18), BM2 (SEQ ID NO:19), HCV TME1 (SEQ ID NQ:20), HCV TME2 (SEQ ID NO:21) and variants thereof having sequence identity to at least one of the sequences of SEQ ID NO: 1-21 of at least 90%. (Claim 12), wherein step d) is carried out at most 120 minutes after step c). (Claim 13) wherein the cell is a prokaryotic microorganism (Claim 16), a kit for carrying out the screening method according to claim 1, the kit comprising: i. DNA encoding at least two membrane proteins and DNA encoding a sensor protein, or ii. DNA encoding at least two sensor proteins and DNA encoding a membrane protein. (Claim 17) are conventional and part of the data gathering process used in the recited judicial exceptions (see MPEP 2106.05(g)). Evidence for conventionality is shown by Carter et al. (Carter et al. Fluorescent Sensors for Measuring Metal Ions in Living Systems. Chemical Reviews 2014, 114 (8), 4564–4601.) which is a review about fluorescent sensors for measuring metal ions in living systems and teaches the largest category of genetically encoded sensors is comprised of protein-based probes that utilize one or more fluorescent proteins (FP) as the fluorophore. The sensors also contain a peptide or protein moiety that serves as a metal binding domain. For single FP-based sensors, metal binding induces a change in the chemical or electronic environment around the chromophore, causing either a change in intensity or a shift in the excitation or emission spectrum. (pg. 4569, col. 1 paragraph 1-3). The dependent claims just limit the analytes for the conventional data gathering of spectroscopy to detect ion levels in and around individual cells. The additional elements of determining the DNA sequence encoding the membrane protein and/or the DNA sequence encoding the sensor protein. (Claim 2), wherein steps a) to f) are carried out for at least 5 membrane proteins with different amino acid sequences (Claim 5), wherein steps a) to f) are carried out for at least 5 sensor proteins with different amino acid sequences. (Claim 6), wherein steps a) to f) are carried out for at least 20 membrane proteins with different amino acid sequences. (Claim 18), wherein steps a) to f) are carried out for at least 50 membrane proteins with different amino acid sequences. (Claim 19), wherein steps a) to f) are carried out for at least 10 2 to 10 8 membrane proteins with different amino acid sequences (Claim 20) are conventional and part of the data gathering process used in the recited judicial exceptions (see MPEP 2106.05(g)). The dependent claims just limit the analytes for the conventional data gathering of method of sequencing. Evidence for conventionality is shown by Afik et al. (Afik et al. Targeted Reconstruction of T Cell Receptor Sequence from Single Cell RNA-Seq Links CDR3 Length to T Cell Differentiation State. Nucleic Acids Research 2017, 45 (16), e148–e148.) which is a discussion about different commercially available single cell sequencing technologies that simultaneously collect membrane protein sequences and cell sequences (pg. 148, col. 1, paragraph 4). Claims 14-15 do not recite any elements in addition to the judicial exception. Therefore, when taken alone, all additional elements in claims 1-13 and 16-20 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 1-20 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 07-06 AIA 15-10-15 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. 07-20-aia AIA 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. 07-23-aia AIA 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. 07-20-02-aia AIA 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. 07-21-aia AIA Claim s 1-14 and 16-20 are rejected under 35 U.S.C. 103 as being unpatentable over Litzlbauer et al. ( Litzlbauer et al. Large Scale Bacterial Colony Screening of Diversified FRET Biosensors. PLOS ONE 2015 , 10 (6), e0119860) in further view of Domínguez et al. (Domínguez, D. C. Calcium Signaling in Prokaryotes ; IntechOpen, 2018) in further view of Pang et al. (Pang et al. Mutational Analysis of the S 21 Pinholin. Molecular Microbiology 2010 , 76 (1), 68–77.). The italicized text corresponds to the instant claim limitations . With respect to the limitations of Claim 1 , Litzlbauer et al. teaches that bacterial calcium under resting conditions is kept low in order to minimize calcium-induced toxicity. Thus, basal FRET measurements under these conditions provides a reasonable read-out of Rmin for calcium sensors (pg. 5, paragraph 2, b) detecting a signal that is dependent on the concentration of the reporter substance in the cell and that is generated with the aid of a sensor protein expressed in the cell (Claim 1)). Litzlbauer et al. also teaches that in order to measure Rmin and Rmax of a given sensor it is necessary to monitor the sensors in both ligand free and ligand bound conditions. Bacterial calcium under resting conditions is kept low in order to minimize calcium-induced toxicity. Thus, basal FRET measurements under these conditions provides a reasonable read-out of Rmin for calcium sensors. In order to obtain Rmax, it was necessary to open up the bacterial cell walls for calcium to penetrate and bind the sensor variants (pg. 5, paragraph 2) and that therefore techniques are necessary that allow recording fluorescence of large numbers of sensor variants both in the ligand free (the minimal ratio Rmin) and ligand bound (the maximal ratio Rmax) state and retrieving the sensors that show the largest FRET change after binding of ligand. Bacterial colony screening is a cost-effective means to screen large numbers of biosensor variants in an acceptable time. E. coli strains can be transformed at high rates, and each bacterial colony expresses but a single sensor variant. On a single agar plate of 10 cm diameter, up to one thousand bacterial colonies each expressing a different sensor variant can be grown side by side, distinguished and imaged using suitable wide-field optics. Moreover, DNA coding for interesting sensors can be easily isolated from bacteria for further analysis, be it sequence analysis, recombinant protein purification for in vitro analysis or subcloning for expression in mammalian cells. The FRET Ca 2+ sensor protein is used to detect the level of Ca 2+ (pg. 2, paragraph 1, d) detecting the signal that is dependent on the concentration of the reporter substance in the cell and that is generated with the aid of the sensor protein expressed in the cell (Claim 1)). Litzlbauer et al. also teaches that data analysis was carried out by a custom written program in Python, which identified the 1–2% best performing colonies on a plate and their position for subsequent picking. Alternatively, R0 of each sensor was plotted against its ΔR/R in Origin 8.1., and new sensor variants were compared to their parental sensors (pg. 3, paragraph 4) and that therefore techniques are necessary that allow recording fluorescence of large numbers of sensor variants both in the ligand free (the minimal ratio Rmin) and ligand bound (the maximal ratio Rmax) state and retrieving the sensors that show the largest FRET change after binding of ligand. Bacterial colony screening is a cost-effective means to screen large numbers of biosensor variants in an acceptable time. E. coli strains can be transformed at high rates, and each bacterial colony expresses but a single sensor variant. On a single agar plate of 10 cm diameter, up to one thousand bacterial colonies each expressing a different sensor variant can be grown side by side, distinguished and imaged using suitable wide-field optics. Moreover, DNA coding for interesting sensors can be easily isolated from bacteria for further analysis, be it sequence analysis, recombinant protein purification for in vitro analysis or subcloning for expression in mammalian cells. The FRET Ca 2+ sensor protein is used to detect the level of Ca 2+ (pg. 2, paragraph 1, e) calculating one or more comparative parameters from the signal strengths detected in steps b) and d), wherein step b) is carried out before step c) and wherein step d) is carried out after step c), (Claim 1)) Litzlbauer et al. also teaches that E. coli strains can be transformed at high rates, and each bacterial colony expresses but a single sensor variant. On a single agar plate of 10 cm diameter, up to one thousand bacterial colonies each expressing a different sensor variant can be grown side by side, distinguished and imaged using suitable wide-field optics. Moreover, DNA coding for interesting sensors can be easily isolated from bacteria for further analysis, be it sequence analysis, recombinant protein purification for in vitro analysis or subcloning for expression in mammalian cells. The FRET Ca 2+ sensor protein is used to detect the level of Ca 2+ and E. Coli colonies are screened for multiple different sensors (pg. 2, paragraph 1, wherein steps a) to e) are carried out for at least two membrane proteins having different amino acid sequences and/or for at least two sensor proteins having different amino acid sequences) With respect to the limitations of Claim 2 , Litzlbauer et al. teaches that DNA coding for interesting sensors can be easily isolated from bacteria for further analysis, be it sequence analysis (pg. 2, paragraph 1, comprising the further step of: f) determining the DNA sequence encoding the membrane protein and/or the DNA sequence encoding the sensor protein (Claim 2)) With respect to the limitations of Claim 3 , Litzlbauer et al. teaches that E. coli strains can be transformed at high rates, and each bacterial colony expresses but a single sensor variant. On a single agar plate of 10 cm diameter, up to one thousand bacterial colonies each expressing a different sensor variant can be grown side by side, distinguished and imaged using suitable wide-field optics. Moreover, DNA coding for interesting sensors can be easily isolated from bacteria for further analysis, be it sequence analysis, recombinant protein purification for in vitro analysis or subcloning for expression in mammalian cells. The FRET Ca 2+ sensor protein is used to detect the level of Ca 2+ and E. Coli colonies are screened for multiple different sensors (pg. 2, paragraph 1, wherein the cell is a microorganism (Claim 3)). With respect to the limitations of Claim 4 , Litzlbauer et al. teaches a method of measuring Ca 2+ levels in E. coli which go through the inner membrane. (pg. 2, paragraph 1, wherein the cell is Escherichia coli, and the cell membrane is the inner membrane of E. coli. (Claim 4)). With respect to the limitations of Claim 6 , Litzlbauer et al. teaches that have been able to identify four of the five best sensors , as determined in vitro, by only picking the best 15 colonies (approximately 2.5% of colonies on the plate) on the blotting paper, thereby reducing the number of variants to test considerably. Using this protocol we were able to enhance FRET ratio change in a minimal domain calcium sensor. Each sensor is a protein with a different sequence (pg. 11, paragraph 2, wherein steps a) to f) are carried out for at least 5 sensor proteins with different amino acid sequences (Claim 6)). With respect to the limitations of Claim 7 , Litzlbauer et al. teaches that therefore techniques are necessary that allow recording fluorescence of large numbers of sensor variants both in the ligand free (the minimal ratio Rmin) and ligand bound (the maximal ratio Rmax) state and retrieving the sensors that show the largest FRET change after binding of ligand. Bacterial colony screening is a cost-effective means to screen large numbers of biosensor variants in an acceptable time. E. coli strains can be transformed at high rates, and each bacterial colony expresses but a single sensor variant. On a single agar plate of 10 cm diameter, up to one thousand bacterial colonies each expressing a different sensor variant can be grown side by side, distinguished and imaged using suitable wide-field optics. Moreover, DNA coding for interesting sensors can be easily isolated from bacteria for further analysis, be it sequence analysis, recombinant protein purification for in vitro analysis or subcloning for expression in mammalian cells. The FRET Ca 2+ sensor protein is used to detect the level of Ca 2+ (pg. 2, paragraph 1, wherein the signal is selected from one or more of a fluorescence signal, a bioluminescence signaler-and an absorption signal (Claim 7)). With respect to the limitations of Claim 8 , Litzlbauer et al. teaches that therefore techniques are necessary that allow recording fluorescence of large numbers of sensor variants both in the ligand free (the minimal ratio Rmin) and ligand bound (the maximal ratio Rmax) state and retrieving the sensors that show the largest FRET change after binding of ligand. Bacterial colony screening is a cost-effective means to screen large numbers of biosensor variants in an acceptable time. E. coli strains can be transformed at high rates, and each bacterial colony expresses but a single sensor variant. On a single agar plate of 10 cm diameter, up to one thousand bacterial colonies each expressing a different sensor variant can be grown side by side, distinguished and imaged using suitable wide-field optics. Moreover, DNA coding for interesting sensors can be easily isolated from bacteria for further analysis, be it sequence analysis, recombinant protein purification for in vitro analysis or subcloning for expression in mammalian cells. The FRET Ca 2+ sensor protein is used to detect the level of Ca 2+ (pg. 2, paragraph 1, wherein the sensor protein is selected from the group of Ca 2 +-specific protein sensors and L-Glu-specific protein sensors (Claim 8)). With respect to the limitations of Claim 14 , Litzlbauer et al. teaches that in order to measure Rmin and Rmax of a given sensor it is necessary to monitor the sensors in both ligand free and ligand bound conditions and that R0 of each sensor was plotted against its ΔR/R in Origin 8.1., and new sensor variants were compared to their parental sensors. Repeated fluorescent measurements are taken to measure calcium intake. Origin 8.1 calculates half-maximum time and the maximum slope. (pg. 3, paragraph 3-5 and pg. 5, paragraph 2-3, wherein step d) is carried out at least five times, and the calculation of the comparative parameter from the signal strengths according to step e) includes empirically fitting the data obtained on the signal strengths with a sigmoid function, wherein the comparative parameter calculated from the signal strengths is the half-maximum time c and/or the maximum slope d. (Claim 14) With respect to the limitations of Claim 16 , Litzlbauer et al. teaches a method of measuring Ca 2+ levels in E. coli which go through the inner membrane. (pg. 2, paragraph 1, wherein the cell is a prokaryotic microorganism (claim 16) With respect to the limitations of Claim 17 , Litzlbauer et al. teaches that data analysis was carried out by a custom written program in Python, which identified the 1–2% best performing colonies on a plate and their position for subsequent picking. Alternatively, R0 of each sensor was plotted against its ΔR/R in Origin 8.1., and new sensor variants were compared to their parental sensors (pg. 3, paragraph 4) and that therefore techniques are necessary that allow recording fluorescence of large numbers of sensor variants both in the ligand free (the minimal ratio Rmin) and ligand bound (the maximal ratio Rmax) state and retrieving the sensors that show the largest FRET change after binding of ligand. Bacterial colony screening is a cost-effective means to screen large numbers of biosensor variants in an acceptable time. E. coli strains can be transformed at high rates, and each bacterial colony expresses but a single sensor variant. On a single agar plate of 10 cm diameter, up to one thousand bacterial colonies each expressing a different sensor variant can be grown side by side, distinguished and imaged using suitable wide-field optics. Moreover, DNA coding for interesting sensors can be easily isolated from bacteria for further analysis, be it sequence analysis, recombinant protein purification for in vitro analysis or subcloning for expression in mammalian cells. The FRET Ca 2+ sensor protein is used to detect the level of Ca 2+ The method includes all of the components of the kit. (pg. 2, paragraph 1, a kit for carrying out the screening method according to claim 1, the kit comprising: i. DNA encoding at least two membrane proteins and DNA encoding a sensor protein, or ii. DNA encoding at least two sensor proteins and DNA encoding a membrane protein (Claim 17)). With respect to the limitations of Claim 18, 19, 20 , Litzlbauer et al. teaches that data analysis was carried out by a custom written program in Python, which identified the 1–2% best performing colonies on a plate and their position for subsequent picking. Alternatively, R0 of each sensor was plotted against its ΔR/R in Origin 8.1., and new sensor variants were compared to their parental sensors (pg. 3, paragraph 4) and that therefore techniques are necessary that allow recording fluorescence of large numbers of sensor variants both in the ligand free (the minimal ratio Rmin) and ligand bound (the maximal ratio Rmax) state and retrieving the sensors that show the largest FRET change after binding of ligand. Bacterial colony screening is a cost-effective means to screen large numbers of biosensor variants in an acceptable time. E. coli strains can be transformed at high rates, and each bacterial colony expresses but a single sensor variant. On a single agar plate of 10 cm diameter, up to one thousand bacterial colonies each expressing a different sensor variant can be grown side by side, distinguished and imaged using suitable wide-field optics. Moreover, DNA coding for interesting sensors can be easily isolated from bacteria for further analysis, be it sequence analysis, recombinant protein purification for in vitro analysis or subcloning for expression in mammalian cells. The FRET Ca 2+ sensor protein is used to detect the level of Ca 2+ (pg. 2, paragraph 1, wherein steps a) to f) are carried out for at least 20 membrane proteins with different amino acid sequences (Claim 18), wherein steps a) to f) are carried out for at least 50 membrane proteins with different amino acid sequences (Claim 19), wherein steps a) to f) are carried out for at least 10 2 to 10 8 membrane proteins with different amino acid sequences (Claim 20). Litzlbauer et al. does not explicitly teach a screening method comprising the steps of: a) providing a cell and a medium surrounding the cell, - wherein the cell has a cell membrane impermeable to a reporter substance,- wherein the quotient of the concentration of the reporter substance in the interior of the cell and the concentration of the reporter substance in the medium surrounding the cell is at least 2 or at most 0.5 (Claim 1) wherein the reporter substance is selected from the group consisting of cations and amino acids (Claim 9) inducing the expression of a membrane protein that increases the permeability of the cell membrane to the reporter substance (Claim 1) wherein steps a) to f) are carried out for at least 5, membrane proteins with different amino acid sequences (Claim 5) wherein the membrane protein is selected from the group consisting of pore-forming membrane proteins and membrane transporters (Claim 10) wherein the membrane protein is selected from the group consisting of holins, pinholins, and ion channels (Claim 11) wherein the membrane protein is selected from the group consisting of S 21 -68 (SEQ ID NO:1), S 21 -71 (SEQ ID NO:2), S 21 -71 M4A (SEQ ID NO:3), SGS ΔTMD1S 21 68 (SEQ ID NO:4), TVMV ΔTMD1-S 21 68 (SEQ ID NO:5), S 105 (SEQ ID NO:6), S 107 (SEQ ID NO:7), S 107 -M3A (SEQ ID NO:8), T4 pinholin (SEQ ID NO:9), T4 ΔC-Tail (SEQ ID NO:10), ΔN-Tail T4 ΔC-Tail (SEQ ID NO:11), K CV NTS (SEQ ID NO: 2), K CV NTS G77S (SEQ ID NO:13), K CV PBCV1 (SEQ ID NO:14), HokB (SEQ ID NO:15), TisB (SEQ ID NO:16), αHLA (SEQ ID NO:17), cWZA (SEQ ID NO:18), BM2 (SEQ ID NO:19), HCV TME1 (SEQ ID NQ:20), HCV TME2 (SEQ ID NO:21) and variants thereof having sequence identity to at least one of the sequences of SEQ ID NO: 1-21 of at least 90%. (Claim 12) wherein step d) is carried out at most 120 minutes after step c) (Claim 13) With respect to the limitations of Claim 1 , Domínguez et al. teaches that all bacteria tested maintained very low levels of cytosolic free Ca2+, even in the presence of 1–10 mM extracellular Ca 2+ . Cytosolic free Ca 2+ in bacterial cells ranges from 100 to 300 nM, very similar values to those observed in eukaryotic cells (pg. 91, paragraph 3, a screening method comprising the steps of: a) providing a cell and a medium surrounding the cell, - wherein the cell has a cell membrane impermeable to a reporter substance,- wherein the quotient of the concentration of the reporter substance in the interior of the cell and the concentration of the reporter substance in the medium surrounding the cell is at least 2 or at most 0.5 (Claim 1)) With respect to the limitations of Claim 9 , Domínguez et al. teaches that all bacteria tested maintained very low levels of cytosolic free Ca2+, even in the presence of 1–10 mM extracellular Ca 2+ . Cytosolic free Ca 2+ in bacterial cells ranges from 100 to 300 nM, very similar values to those observed in eukaryotic cells (pg. 91, paragraph 3, wherein the reporter substance is selected from the group consisting of cations and amino acids (Claim 9)). With respect to the limitations of Claim 1 , Pang et al. teaches that pinholins expression forms holes in the pores the fact that they form small holes. This would increase calcium permeability. (pg. 68, col. 2, Introduction, paragraph 1, inducing the expression of a membrane protein that increases the permeability of the cell membrane to the reporter substance (Claim 1)). With respect to the limitations of Claim 5 , Pang et al. teaches multiple different variations of the pinholin membrane protein (table 1, pg. 71, ( wherein steps a) to f) are carried out for at least 5, membrane proteins with different amino acid sequences (Claim 5)). With respect to the limitations of Claim 10 , Pang et al. teaches that pinholins expression forms holes in the pores the fact that they form small holes. This would increase calcium permeability. (pg. 68, col. 2, Introduction, paragraph 1, wherein the membrane protein is selected from the group consisting of pore-forming membrane proteins and membrane transporters (Claim 10)). With respect to the limitations of Claim 11 , Pang et al. teaches that pinholins expression forms holes in the pores the fact that they form small holes. This would increase calcium permeability. (pg. 68, col. 2, Introduction, paragraph 1, wherein the membrane protein is selected from the group consisting of holins, pinholins, and ion channels (Claim 11)). With respect to the limitations of Claim 12 , Pang et al. teaches multiple different variations of the pinholin membrane protein including S 21 -68 (pg. 70, col. 1, paragraph 1 and table 1, pg. 71, wherein the membrane protein is selected from the group consisting of S 21 -68 (SEQ ID NO:1), S 21 -71 (SEQ ID NO:2), S 21 -71 M4A (SEQ ID NO:3), SGS ΔTMD1S 21 68 (SEQ ID NO:4), TVMV ΔTMD1-S 21 68 (SEQ ID NO:5), S 105 (SEQ ID NO:6), S 107 (SEQ ID NO:7), S 107 -M3A (SEQ ID NO:8), T4 pinholin (SEQ ID NO:9), T4 ΔC-Tail (SEQ ID NO:10), ΔN-Tail T4 ΔC-Tail (SEQ ID NO:11), K CV NTS (SEQ ID NO: 2), K CV NTS G77S (SEQ ID NO:13), K CV PBCV1 (SEQ ID NO:14), HokB (SEQ ID NO:15), TisB (SEQ ID NO:16), αHLA (SEQ ID NO:17), cWZA (SEQ ID NO:18), BM2 (SEQ ID NO:19), HCV TME1 (SEQ ID NQ:20), HCV TME2 (SEQ ID NO:21) and variants thereof having sequence identity to at least one of the sequences of SEQ ID NO: 1-21 of at least 90%. (Claim 12)). With respect to the limitations of Claim 13 , Pang et al. teaches a triggering time of 50 minutes after deduction (pg. 76, Table 1, wherein step d) is carried out at most 120 minutes after step c) (Claim 13)). A person having ordinary skill in the art would be motivated to combine the methods of Domínguez et al. with the methods of Litzlbauer et al. because Litzlbauer et al. is a method of large-scale bacterial colony screening of diversified FRET biosensors and Domínguez et al. is a review of calcium signaling in prokaryotes and thus both works are in the same field of endeavor and therefore a person of ordinary skill in the art would be motivated to combine the prior art. A person having ordinary skill in the art would also be motivated to introduce the methods of Pang et al. in order to specifically target the pinholin protein. There is a reasonable expectation of success because known methods are being combined in a comprehensive workflow where each individual portion does not change and therefore it is expected to work and yield predictable results . 07-21-aia AIA Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Litzlbauer et al. in further view of Domínguez et al. in further view of Pang et al. as applied to claims 1-14 and 16-20 above in further view of Venegas et al. (Venegas et al. A Comprehensive Equation for the Pulmonary Pressure- Volume Curve. Journal of Applied Physiology 1998 , 84 (1), 389–395.) The italicized text corresponds to the instant claim limitations . The limitations of claims 1-14 and 16-20 have been taught by Litzlbauer et al. in further view of Domínguez et al. in further view of Pang et al. Litzlbauer et al. in further view of Domínguez et al. in further view of Pang et al. does not explicitly teach wherein the data obtained on the signal strengths are fitted empirically with the following equation 1 PNG media_image1.png 127 517 media_image1.png Greyscale (claim 15) However, these limitations were known in the art at the time of the effective filing date of the invention, as taught by Venegas et al. With respect to the limitations of claim 15, Venegas et al. teaches the equation PNG media_image2.png 150 409 media_image2.png Greyscale for fitting a pulmonary pressure-volume curve which is sigmoidal. (pg. 390, col. 1, paragraph 3, equation 1, wherein the data obtained on the signal strengths are fitted empirically with the following equation 1 PNG media_image1.png 127 517 media_image1.png Greyscale (claim 15) ) A person having ordinary skill in the art would be motivated to combine the method of method for screening pore-forming membrane proteins, membrane transporters and molecular switches taught by Litzlbauer et al. in further view of Domínguez et al. in further view of Pang et al. with the sigmoidal curve equation of Venegas et al. because the formula is a known technique for fitting sigmoidal curves and therefore a person of ordinary skill in the art would be motivated to look at previous methods to fit sigmoidal curves. There is a reasonable expectation of success because known methods to fit a curveare being applied where each individual portion does not change and therefore it is expected to work and yield predictable results. Conclusion Any inquiry concerning this communication or earlier communications from the examiner should be directed to Connor Beveridge whose telephone number is 571-272-2099. The examiner can normally be reached Monday - Thursday 9 am - 5 pm. Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Karlheinz Skowronek can be reached at 571-272-9047. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300. Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000. /C.H.B./Examiner, Art Unit 1687 /Karlheinz R. Skowronek/Supervisory Patent Examiner, Art Unit 1687 Application/Control Number: 17/641,835 Page 2 Art Unit: 1687 Application/Control Number: 17/641,835 Page 3 Art Unit: 1687 Application/Control Number: 17/641,835 Page 4 Art Unit: 1687 Application/Control Number: 17/641,835 Page 5 Art Unit: 1687 Application/Control Number: 17/641,835 Page 6 Art Unit: 1687 Application/Control Number: 17/641,835 Page 7 Art Unit: 1687 Application/Control Number: 17/641,835 Page 8 Art Unit: 1687 Application/Control Number: 17/641,835 Page 9 Art Unit: 1687 Application/Control Number: 17/641,835 Page 10 Art Unit: 1687 Application/Control Number: 17/641,835 Page 11 Art Unit: 1687 Application/Control Number: 17/641,835 Page 12 Art Unit: 1687 Application/Control Number: 17/641,835 Page 13 Art Unit: 1687 Application/Control Number: 17/641,835 Page 14 Art Unit: 1687 Application/Control Number: 17/641,835 Page 15 Art Unit: 1687 Application/Control Number: 17/641,835 Page 16 Art Unit: 1687 Application/Control Number: 17/641,835 Page 17 Art Unit: 1687 Application/Control Number: 17/641,835 Page 18 Art Unit: 1687 Application/Control Number: 17/641,835 Page 19 Art Unit: 1687 Application/Control Number: 17/641,835 Page 20 Art Unit: 1687 Application/Control Number: 17/641,835 Page 21 Art Unit: 1687 Application/Control Number: 17/641,835 Page 22 Art Unit: 1687 Application/Control Number: 17/641,835 Page 23 Art Unit: 1687 Application/Control Number: 17/641,835 Page 24 Art Unit: 1687 Application/Control Number: 17/641,835 Page 25 Art Unit: 1687