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 .
Priority
The instant application filed on 01/30/2024 claims priority to TW112148039 filed on 12/11/2023. The certified copy of TW112148039 is not in English; therefore, the effective filing date of the instantly claimed invention is 01/30/2024.
Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 09/01/2024, 03/28/2025, 07/17/2025 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Claim Rejections - 35 USC § 103, Obviousness
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 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.
Claims 1-4, 6, and 10 are rejected under 35 U.S.C. 103 as being unpatentable over Tillich (The Optimal Mutagen Dosage to Induce Point-Mutations in Synechocystis sp. PCC6803 and Its Application to Promote Temperature; 2012 – cited in the IDS filed on 09/01/2024) in view of Trovao (Random Mutagenesis as a Promising Tool for Microalgal Strain Improvement towards Industrial Production; 2022 – cited in the IDS filed on 09/01/2024).
Tillich’s general disclosure relates to characterizing “the lethality and rate of non-lethal point mutations for ultraviolet radiation and methyl methanesulphonate on the model cyanobacteria Synechocystis sp. PCC6803” (see, e.g., Tillich, abstract). Additionally, Tillich discloses “After four distinct rounds of treatment (two with each mutagen) the temperature tolerance of the strain was effectively raised by about 2°C. Coupled with an appropriate in vivo screening, the described methods should be applicable to induce a variety of desirable characteristics in various strains” (see, e.g., Tillich, abstract).
Regarding claim 1 pertaining to the method of cultivating cyanobacteria, Tillich teaches a model cyanobacteria Synechocystis sp. PCC6803 (see, e.g., Tillich, abstract), wherein the cyanobacteria undergoes four rounds of mutagenesis by UV radiation (i.e., physical mutagenesis) and methyl methanesulphonate (MMS) (i.e., chemical mutagenesis), wherein the UV radiation mutagenesis rounds are performed before the MMS chemical mutagenesis rounds (see, e.g., Tillich, abstract & Table 1). The mutants obtained after the UV radiation steps were subsequently exposed to MMS (see, e.g., Tillich, Table 1); therefore, one of ordinary skill in the art would understand that the primary mutant cyanobacteria obtained from UV radiation is exposed to MMS to produce secondary mutant cyanobacteria. Tillich teaches that the mutant cyanobacteria were subjected to a temperature tolerance selection (see, e.g., Tillich, Temperature Tolerance Selection”, pg. 3 & “Applied Mutagenesis”, pg. 3). Tillich teaches that the physical mutagenesis fatality rate is 50-80% at a UV dosage of 50-75 J/m2 (see, e.g., Tillich, Figure 1), and that the chemical mutagenesis fatality rate is 40-55% at an MMS dosage less than 1 vol% (see, e.g., Tillich, Figure 2).
Regarding claims 2-3 pertaining to the fatality rate, Tillich teaches that the physical mutagenesis fatality rate is 50-80% at a UV dosage of 50-75 J/m2 (see, e.g., Tillich, Figure 1), and that the chemical mutagenesis fatality rate is 40-55% at an MMS dosage less than 1 vol% (see, e.g., Tillich, Figure 2).
Regarding claim 4 pertaining to the physical mutagenesis UV source, Tillich teaches that a Synechocystis culture was irradiated up to 300 J/m2 and that the time of irradiation was automatically adapted by the devices integrated sensor (see, e.g., Tillich, “UV Mutagenesis”, pg. 3). One of ordinary skill in the art would be able to calculate the illuminance (i.e., mW/cm2) from the irradiation dose (i.e., J/m2) based on the time of exposure.
Regarding claim 6 pertaining to the temperature resistance test, Tillich teaches that the mutant cyanobacteria were exposed to, and able to stably grow at, temperatures around 45oC (see, e.g., Tillich, “Applied Mutagenesis”, pg. 4).
Regarding claim 10 pertaining to subjecting the cyanobacteria to chemical mutagenesis again, Tillich teaches two UV exposure steps followed by two MMS exposure steps; therefore, after the first MMS exposure, the cyanobacteria is exposed to MMS again (see, e.g., Tillich, Table 1).
However, Tillich does not teach: an environmental resistance test, so as to obtain target cyanobacteria (claim 1).
Trovao’s general disclosure relates to a review discussing “different microalgal strain improvement approaches and their applications, with a primary focus on random mutagenesis” (see, e.g., Trovao, abstract). Additionally, Trovao discloses adaptive laboratory techniques, wherein “Adaptive laboratory evolution consists of exposing microalgae to specific stress conditions (e.g., high salinity, CO2, glucose, or flue gas concentration) during prolonged periods (months or years) to promote the selection of spontaneous mutations that confer an adaptive advantage to the growth conditions (Figure 5). Usually, in adaptive laboratory evolution experiments, the mutations detected have been mapped to stress-induced genes. Under stressful conditions, the stress-induced genes are activated at the expense of housekeeping genes and growth. If the stressful conditions are withdrawn, the stress-induced genes are repressed and the cell resumes its normal activity. The conditions of adaptive laboratory evolution keep the stress constant from one generation to the next and the stress response is kept active, so that any mutation that enables the cell to grow under stressful conditions is likely to be favored. Likewise, each generational cycle improves the original wildtype strain, selecting cells with higher environmental tolerance, and thus, displaying more robust, tailor-made phenotypes” (see, e.g., Trovao, Section 2.2, pg. 12).
Regarding claim 1 pertaining to the environmental resistance test, Trovao teaches that generated mutants can be tested and selected for by exposure to environmental stresses, such as extreme salinities or temperatures, light or dark conditions, CO2 levels, or nutrient stress (see, e.g., Trovao, Section 2.1.3, pg. 9).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce Tillich’s mutant Synechocystis sp. PCC6803 cyanobacteria, wherein the mutant Synechocystis sp. PCC6803 is tested and selected for by exposure to environmental stresses, as taught by Trovao. One would have been motivated to do so because Trovao teaches that exposure of the newly generated mutants to environmental stresses is a more direct approach to selecting for mutants compared to selection based on a desired phenotype (see, e.g., Trovao, Section 2.1.3, pgs. 9-10). Additionally, Trovao teaches selection of mutants by exposure to environmental stressors, such as high salinity, high temperature, and/or high pH leads to the generation of salt-resistant, thermotolerant, and alkali-resistant strains (see, e.g., Trovao, Section 2.1.3, pg. 10). Moreover, Tillich teaches the generation of Synechocystis sp. PCC6803 mutants through exposure to UV (i.e., physical exposure) and MMS (i.e., chemical exposure); therefore, based on these teachings, it would have been obvious to produce Synechocystis sp. PCC6803 cyanobacteria mutants that have been selected for by exposure to high temperatures and environmental stresses in order to produce mutants that are thermotolerant and resistant to other environmental stresses, such as salt-resistance and alkali-resistance. One would have expected success because Tillich and Trovao both teach generation of mutant bacteria following random mutagenesis via chemical and physical means.
Regarding claim 4’s UV irradiation time limitations, those working in the biological and/or pharmaceutical arts would understand that the adjustments of particular conventional working conditions (e.g., time of exposure) is deemed a matter of judicious selection and routine optimization, which is within the purview of the skilled artisan. For example, Tillich teaches that a Synechocystis culture was irradiated up to 300 J/m2 and that the time of irradiation was automatically adapted by the devices integrated sensor (see, e.g., Tillich, “UV Mutagenesis”, pg. 3). Furthermore, based on the teachings of Tillich, one of ordinary skill in the art would readily understand that manipulating the amount of time the Synechocystis culture was irradiated would influence the survival rate of the culture and the number of mutants generated (see, e.g., Tillich, Figure 1 & whole document). This is motivation for one of ordinary skill in the art to practice or test the parameter widely to find those that are functional or optimal which then would be inclusive or cover the steps as instantly claimed. Absent any teaching of criticality by the Applicant concerning the amount of time for UV irradiation, it would be prima facie obvious that one of ordinary skill in the art would recognize that this limitation is a result effective variable which can be met as a matter of routine optimization.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Tillich and Trovao as applied to claims 1-4, 6, and 10 above, and further in view of Mishra (Mutagenesis and Isolation of Morphological Mutants Impaired in Nitrogen-Fixing Capacity from a Cyanobacterium Gloeotrichia ghosei; 1986 – cited in the IDS filed on 09/01/2024).
The references of Tillich and Trovao are discussed above.
However, the references do not teach: wherein nitrosoguanidine (NTG) having a concentration of between 50 pg/ml and 300 g/ml is used in the chemical mutagenesis for a processing time within a range between 0.5 minutes and 2 minutes (claim 5).
Mishra’s general disclosure relates to “Ultraviolet (u.v.) irradiation and treatment with nitrosoguanidine (NTG) were used to produce mutants of the cyanobacterium Gloeotrichia ghosei”, wherein “three non-nitrogen fixing (nif-) mutants, which were also altered in their morphological characteristics, were isolated (see, e.g., Mishra, abstract).
Regarding claim 5 pertaining to NTG, Mishra teaches exposure of G. ghosei to NTG at a concentration of 100 µg/mL and samples were withdrawn at 15 min intervals (see, e.g., Mishra, “NTG-sensitivity”, pg. 70).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce Tillich and Trovao’s mutant Synechocystis sp. PCC6803 cyanobacteria by physical and chemical exposure, wherein the chemical exposure is NTG, as taught by Mishra. One would have been motivated to do so because Mishra teaches generation and isolation of G. ghosei mutants following NTG exposure (see, e.g., Mishra, pg. 71). Moreover, Tillich teaches chemical MMS exposure of mutant Synechocystis sp. PCC6803 cyanobacteria in order to generate mutants followed by assessing survival rates to the exposed mutant Synechocystis sp. PCC6803 cyanobacteria (see, e.g., Tillich, MMS mutagenesis, pg. 3 & Figure 2). Therefore, MMS and NTG are chemical agents both used to generate mutant cyanobacteria. Trovao teaches that NTG and MMS are alkylating chemical mutagen agents that substitutes a hydrogen ion for an alkyl group on a DNA base (see, e.g., Trovao, Section 2.1.2). Additionally, Trovao teaches that NTG and MMS are used in microalgal strain improvement and “trigger a similar chemical mutagenesis mechanism in DNA, which enables high-frequency point mutation and emergence of novel phenotypes” (see, e.g., Trovao, Section 2.1.2). MMS and NTG are art recognized equivalents for the same purpose of triggering chemical mutagenesis in DNA by high-frequency point mutations, and MMS and NTG are both alkylating chemical mutagen agents that substitutes a hydrogen ion for an alkyl group on a DNA base; therefore, it would have been obvious to substitute MMS, as taught in Tillich, for NTG, as taught in Mishra and Trovao. One would have expected success because Tillich, Trovao, and Mishra all teach generation of cyanobacteria mutants via physical and chemical exposure.
Regarding claim 5’s NTG exposure time limitations, those working in the biological and/or pharmaceutical arts would understand that the adjustments of particular conventional working conditions (e.g., time of exposure) is deemed a matter of judicious selection and routine optimization, which is within the purview of the skilled artisan. For example, Mishra teaches “From the survival curves, it was found that 30 min treatment of an exponentially growing culture with NTG or 60s with u.v. light gave 5 to 10% survival and the treatments were chosen to induce mutagenesis” (see, e.g., Mishra, NTG sensitivity, pg. 72). Furthermore, based on the teachings of Mishra, one of ordinary skill in the art would readily understand that manipulating the amount of time the Synechocystis culture was exposed to NTG would influence the survival rate of the culture and the number of mutants generated (see, e.g., see, e.g., Mishra, NTG sensitivity, pg. 72). This is motivation for one of ordinary skill in the art to practice or test the parameter widely to find those that are functional or optimal which then would be inclusive or cover the steps as instantly claimed. Absent any teaching of criticality by the Applicant concerning the amount of time for NTG exposure, it would be prima facie obvious that one of ordinary skill in the art would recognize that this limitation is a result effective variable which can be met as a matter of routine optimization.
Claims 7-9 are rejected under 35 U.S.C. 103 as being unpatentable over Tillich and Trovao as applied to claims 1-4, 6, and 10 above, and further in view of Gyure (US 2009/0130704; Date of Publication: May 21, 2009) and Nashimoto (JP 11207122; Date of Publication: August 3, 1999 – cited in the IDS filed on 03/28/2025).
The references of Tillich and Trovao are discussed above.
However, the references do not teach: wherein in the environment resistance test, the initial cyanobacteria are placed in a mixed gas at a ventilation ratio within a range between 0.5% and 9%, so as to filter out the target cyanobacteria (claim 7); or wherein the mixed gas includes hydrogen, acetylene, methane, hydrogen sulfide, and acetaldehyde (claim 8); or wherein the mixed gas includes 30 ppm to 50 ppm of the hydrogen, 150 ppm to 250 ppm of the acetylene, 100 ppm to 200 ppm of the methane, 0.1 ppm to 1 ppm of the hydrogen sulfide, and1 ppm to 5 ppm of the acetaldehyde (claim 9).
Gyure’s general disclosure relates to bioreactors having a selectively permeable porous material for culturing cells and organisms for production of gaseous products, including hydrogen, biomass, chemical and pharmaceuticals (see, e.g., Gyure, abstract).
Regarding claims 7-9 pertaining to the mixed gas, Gyrue teaches inoculating and growing cyanobacteria, such as Synechococcus sp. PCC 7942, in a bioreactor (see, e.g., Gyrue, [0087], [0212]), wherein the bioreactor comprises porous materials that are gas permeable to hydrogen, methane, acetylene, and hydrogen sulfide (see, e.g., Gyrue, [0048]).
Nashimoto’s general disclosure relates to purify polluted air through use of photosynthetic bacteria in a purification vessel, wherein the purification vessel is gas permeable and the photosynthetic bacteria are cyanobacteria (see, e.g., Nashimoto, English Translation, abstract & Embodiment 1).
Regarding claims 7-9 pertaining to the mixed gas, Nashimoto teaches that contaminated air used for purification by cyanobacteria can include aldehydes such as hydrogen sulfide and acetaldehyde, wherein these gases are exposed to the cyanobacteria by passing through a gas permeable membrane (see, e.g., Nashimoto, English Translation, Embodiment 1 & Figures 1-2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce Tillich and Trovao’s mutant Synechocystis sp. PCC6803 cyanobacteria by physical and chemical exposure, wherein the mutant Synechocystis sp. PCC6803 cyanobacteria produced after physical and chemical exposure is subsequently exposed to mixed gas, as taught by Gyrue and Nashimoto. One would have been motivated to do so because Gyrue teaches inoculating and culturing Synechococcus sp. PCC 7942 in a bioreactor, wherein the bioreactor comprises gas permeable porous materials that are permeable to hydrogen, methane, acetylene, and hydrogen sulfide (see, e.g., Gyrue, [0048]) for which the cyanobacteria can be exposed to. Nashimoto teaches a method of air purification using cyanobacteria, such as Synechocystis, and a gas permeable membrane (see, e.g., Nashimoto, English Translation, Abstract & pg. 4). Nashimoto teaches that the cyanobacteria are placed into the vessel, wherein the irradiated cyanobacteria are introduced to aldehydes such as hydrogen sulfide and acetaldehyde (see, e.g., Nashimoto, English Translation, Embodiment 1 & Figures 1-2). Therefore, Gyrue and Nashimoto both teach introduction of cyanobacteria to mixed gases via gas permeable membranes and one would want to expose cyanobacteria to these mixed gases in order to produce mutant cyanobacteria that are resistant to these gases. Trovao teaches “Adaptive laboratory evolution consists of exposing microalgae to specific stress conditions (e.g., high salinity, CO2, glucose, or flue gas concentration)”, wherein “the conditions of adaptive laboratory evolution keep the stress constant from one generation to the next and the stress response is kept active, so that any mutation that enables the cell to grow under stressful conditions is likely to be favored. Likewise, each generational cycle improves the original wildtype strain, selecting cells with higher environmental tolerance, and thus, displaying more robust, tailor-made phenotypes” (see, e.g., Trovao, Section 2.2, pg. 12). Therefore, based on these teachings, the chemical and physical mutagenesis introduced to the cyanobacteria will produce a cyanobacteria mutant with higher environmental tolerance through adaptive laboratory evolution, wherein the cyanobacteria produced have increased tolerance to mixed gases. One would have expected success because Tillich, Trovao, Gyrue, and Nashimoto all teach exposure of cyanobacteria to physical, chemical, or environmental stressors.
Regarding claim 7’s ventilation ratio limitations and claim 9’s ppm ratio limitations, those working in the biological and/or pharmaceutical arts would understand that the adjustments of particular conventional working conditions (e.g., ratios, percentages, concentrations, etc.) is deemed a matter of judicious selection and routine optimization, which is within the purview of the skilled artisan. For example, Gyure teaches growth of Synechococcus sp. PCC 7942 in a bioreactor (see, e.g., Gyrue, [0087], [0212]), wherein the bioreactor comprises porous materials that are gas permeable to hydrogen, methane, acetylene, and hydrogen sulfide (see, e.g., Gyrue, [0048]). Additionally, Gyrue teaches that the porous material has “a pore size of about 1000 nm, 500 nm, or about 200 nm” (see, e.g., Gyrue, [0121]). Based on Gyrue’s teachings, one of ordinary skill in the art would readily understand that the pore size that the gas passes through will influence the percentage, ventilation ratio, and concentration (i.e., ppm) of gas reaching the cyanobacteria. This is motivation for one of ordinary skill in the art to practice or test the parameter widely to find those that are functional or optimal which then would be inclusive or cover the steps as instantly claimed. Absent any teaching of criticality by the Applicant concerning the ventilation ratio, it would be prima facie obvious that one of ordinary skill in the art would recognize that this limitation is a result effective variable which can be met as a matter of routine optimization.
Claims 11-12 and 14-15 are rejected under 35 U.S.C. 103 as being unpatentable over Tillich (The Optimal Mutagen Dosage to Induce Point-Mutations in Synechocystis sp. PCC6803 and Its Application to Promote Temperature; 2012 – cited in the IDS filed on 09/01/2024) in view of Gyure (US 2009/0130704; Date of Publication: May 21, 2009) and Nashimoto (JP 11207122; Date of Publication: August 3, 1999 – cited in the IDS filed on 03/28/2025).
Tillich’s general disclosure is discussed above.
Regarding claim 11 pertaining to the method of cultivating cyanobacteria, Tillich teaches a model cyanobacteria Synechocystis sp. PCC6803 (see, e.g., Tillich, abstract), wherein the cyanobacteria undergoes four rounds of mutagenesis by UV radiation (i.e., physical mutagenesis) and methyl methanesulphonate (MMS) (i.e., chemical mutagenesis), wherein the UV radiation mutagenesis rounds are performed before the MMS chemical mutagenesis rounds (see, e.g., Tillich, abstract & Table 1). The mutants obtained after the UV radiation steps were subsequently exposed to MMS (see, e.g., Tillich, Table 1); therefore, one of ordinary skill in the art would understand that the primary mutant cyanobacteria obtained from UV radiation is exposed to MMS to produce secondary mutant cyanobacteria. Tillich teaches that the physical mutagenesis fatality rate is 50-80% at a UV dosage of 50-75 J/m2 (see, e.g., Tillich, Figure 1), and that the chemical mutagenesis fatality rate is 40-55% at an MMS dosage less than 1 vol% (see, e.g., Tillich, Figure 2). Tillich teaches that a Synechocystis culture was irradiated up to 300 J/m2 and that the time of irradiation was automatically adapted by the devices integrated sensor (see, e.g., Tillich, “UV Mutagenesis”, pg. 3). One of ordinary skill in the art would be able to calculate the illuminance (i.e., mW/cm2) from the irradiation dose (i.e., J/m2) based on the time of exposure. Tillich teaches that the mutant cyanobacteria were subjected to a temperature tolerance selection, wherein mutant cyanobacteria were exposed to, and able to stably grow at, temperatures around 45oC (see, e.g., Tillich, Temperature Tolerance Selection”, pg. 3 & “Applied Mutagenesis”, pg. 3).
Regarding claim 12 pertaining to physical mutagenesis, Tillich teaches that a Synechocystis culture was irradiated up to 300 J/m2 and that the time of irradiation was automatically adapted by the devices integrated sensor (see, e.g., Tillich, “UV Mutagenesis”, pg. 3). One of ordinary skill in the art would be able to calculate the illuminance (i.e., mW/cm2) from the irradiation dose (i.e., J/m2) based on the time of exposure.
Regarding claim 15 pertaining to subjecting the cyanobacteria to chemical mutagenesis again, Tillich teaches two UV exposure steps followed by two MMS exposure steps; therefore, after the first MMS exposure, the cyanobacteria is exposed to MMS again (see, e.g., Tillich, Table 1).
However, Tillich does not teach: placing the initial cyanobacteria in a mixed gas at a ventilation ratio within a range between 0.5% and 9%, so as to filter out target cyanobacteria; wherein the mixed gas is a mixture that includes hydrogen, acetylene, methane, hydrogen sulfide, and acetaldehyde (claim 11); or wherein the mixed gas includes 30 ppm to 50 ppm of the hydrogen, 150 ppm to 250 ppm of the acetylene, 100 ppm to 200 ppm of the methane, 0.1 ppm to 1 ppm of the hydrogen sulfide, and1 ppm to 5 ppm of the acetaldehyde (claim 14).
Gyure’s general disclosure is discussed above.
Regarding claims 11 and 14 pertaining to the mixed gas, Gyrue teaches inoculating cyanobacteria in a bioreactor (see, e.g., Gyrue, [0212]), wherein the bioreactor comprises porous materials that are gas permeable to hydrogen, methane, acetylene, and hydrogen sulfide (see, e.g., Gyrue, [0048]). Therefore, exposure of these gases to cyanobacteria within the bioreactor would inherently result in filtering out the target cyanobacteria.
Nashimoto’s general disclosure is discussed above.
Regarding claims 11 and 14 pertaining to the mixed gas, Nashimoto teaches that contaminated air used for purification by cyanobacteria can include aldehydes such as hydrogen sulfide and acetaldehyde, wherein these gases are exposed to the cyanobacteria by passing through a gas permeable membrane (see, e.g., Nashimoto, English Translation, Embodiment 1 & Figures 1-2). Therefore, exposure of these gases to cyanobacteria within the air purifier device would inherently result in filtering out the target cyanobacteria.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce Tillich and Trovao’s mutant Synechocystis sp. PCC6803 cyanobacteria by physical and chemical exposure, wherein the mutant Synechocystis sp. PCC6803 cyanobacteria produced after physical and chemical exposure is subsequently exposed to mixed gas, as taught by Gyrue and Nashimoto. One would have been motivated to do so because Gyrue teaches inoculating and culturing Synechococcus sp. PCC 7942 in a bioreactor, wherein the bioreactor comprises gas permeable porous materials that are permeable to hydrogen, methane, acetylene, and hydrogen sulfide (see, e.g., Gyrue, [0048]) for which the cyanobacteria can be exposed to. Nashimoto teaches a method of air purification using cyanobacteria, such as Synechocystis, and a gas permeable membrane (see, e.g., Nashimoto, English Translation, Abstract & pg. 4). Nashimoto teaches that the cyanobacteria are placed into the vessel, wherein the irradiated cyanobacteria are introduced to aldehydes such as hydrogen sulfide and acetaldehyde (see, e.g., Nashimoto, English Translation, Embodiment 1 & Figures 1-2). Therefore, Gyrue and Nashimoto both teach introduction of cyanobacteria to mixed gases via gas permeable membranes and one would want to expose cyanobacteria to these mixed gases in order to produce mutant cyanobacteria that are resistant to these gases. Trovao teaches “Adaptive laboratory evolution consists of exposing microalgae to specific stress conditions (e.g., high salinity, CO2, glucose, or flue gas concentration)”, wherein “the conditions of adaptive laboratory evolution keep the stress constant from one generation to the next and the stress response is kept active, so that any mutation that enables the cell to grow under stressful conditions is likely to be favored. Likewise, each generational cycle improves the original wildtype strain, selecting cells with higher environmental tolerance, and thus, displaying more robust, tailor-made phenotypes” (see, e.g., Trovao, Section 2.2, pg. 12). Therefore, based on these teachings, the chemical and physical mutagenesis introduced to the cyanobacteria will produce a cyanobacteria mutant with higher environmental tolerance through adaptive laboratory evolution, wherein the cyanobacteria produced have increased tolerance to mixed gases. One would have expected success because Tillich, Trovao, Gyrue, and Nashimoto all teach exposure of cyanobacteria to physical, chemical, or environmental stressors.
Regarding claim 11’s ventilation ratio limitations and claim 14’s ppm ratio limitations, those working in the biological and/or pharmaceutical arts would understand that the adjustments of particular conventional working conditions (e.g., ratios, percentages, concentrations, etc.) is deemed a matter of judicious selection and routine optimization, which is within the purview of the skilled artisan. For example, Gyure teaches growth of Synechococcus sp. PCC 7942 in a bioreactor (see, e.g., Gyrue, [0087], [0212]), wherein the bioreactor comprises porous materials that are gas permeable to hydrogen, methane, acetylene, and hydrogen sulfide (see, e.g., Gyrue, [0048]). Additionally, Gyrue teaches that the porous material has “a pore size of about 1000 nm, 500 nm, or about 200 nm” (see, e.g., Gyrue, [0121]). Based on Gyrue’s teachings, one of ordinary skill in the art would readily understand that the pore size that the gas passes through will influence the percentage, ventilation ratio, and concentration (i.e., ppm) of gas reaching the cyanobacteria. This is motivation for one of ordinary skill in the art to practice or test the parameter widely to find those that are functional or optimal which then would be inclusive or cover the steps as instantly claimed. Absent any teaching of criticality by the Applicant concerning the ventilation ratio, it would be prima facie obvious that one of ordinary skill in the art would recognize that this limitation is a result effective variable which can be met as a matter of routine optimization.
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Tillich, Gyure, and Nashimoto as applied to claims 11-12 and 14-15 above, and further in view of Mishra (Mutagenesis and Isolation of Morphological Mutants Impaired in Nitrogen-Fixing Capacity from a Cyanobacterium Gloeotrichia ghosei; 1986 – cited in the IDS filed on 09/01/2024).
The references of Tillich and Gyrue, and Nashimoto are discussed above.
However, the references do not teach: wherein nitrosoguanidine (NTG) having a concentration of between 50 pg/ml and 300 g/ml is used in the chemical mutagenesis for a processing time within a range between 0.5 minutes and 2 minutes (claim 13).
Mishra’s general disclosure is discussed above.
Regarding claim 13 pertaining to NTG, Mishra teaches exposure of G. ghosei to NTG at a concentration of 100 µg/mL and samples were withdrawn at 15 min intervals (see, e.g., Mishra, “NTG-sensitivity”, pg. 70).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce Tillich and Trovao’s mutant Synechocystis sp. PCC6803 cyanobacteria by physical and chemical exposure, wherein the chemical exposure is NTG, as taught by Mishra. One would have been motivated to do so because Mishra teaches generation and isolation of G. ghosei mutants following NTG exposure (see, e.g., Mishra, pg. 71). Moreover, Tillich teaches chemical MMS exposure of mutant Synechocystis sp. PCC6803 cyanobacteria in order to generate mutants followed by assessing survival rates to the exposed mutant Synechocystis sp. PCC6803 cyanobacteria (see, e.g., Tillich, MMS mutagenesis, pg. 3 & Figure 2). Therefore, MMS and NTG are chemical agents both used to generate mutant cyanobacteria. Trovao teaches that NTG and MMS are alkylating chemical mutagen agents that substitutes a hydrogen ion for an alkyl group on a DNA base (see, e.g., Trovao, Section 2.1.2). Additionally, Trovao teaches that NTG and MMS are used in microalgal strain improvement and “trigger a similar chemical mutagenesis mechanism in DNA, which enables high-frequency point mutation and emergence of novel phenotypes” (see, e.g., Trovao, Section 2.1.2). MMS and NTG are art recognized equivalents for the same purpose of triggering chemical mutagenesis in DNA by high-frequency point mutations, and MMS and NTG are both alkylating chemical mutagen agents that substitutes a hydrogen ion for an alkyl group on a DNA base; therefore, it would have been obvious to substitute MMS, as taught in Tillich, for NTG, as taught in Mishra and Trovao. One would have expected success because Tillich, Trovao, and Mishra all teach generation of cyanobacteria mutants via physical and chemical exposure.
Regarding claim 13’s NTG exposure time limitations, those working in the biological and/or pharmaceutical arts would understand that the adjustments of particular conventional working conditions (e.g., time of exposure) is deemed a matter of judicious selection and routine optimization, which is within the purview of the skilled artisan. For example, Mishra teaches “From the survival curves, it was found that 30 min treatment of an exponentially growing culture with NTG or 60s with u.v. light gave 5 to 10% survival and the treatments were chosen to induce mutagenesis” (see, e.g., Mishra, NTG sensitivity, pg. 72). Furthermore, based on the teachings of Mishra, one of ordinary skill in the art would readily understand that manipulating the amount of time the Synechocystis culture was exposed to NTG would influence the survival rate of the culture and the number of mutants generated (see, e.g., Mishra, NTG sensitivity, pg. 72). This is motivation for one of ordinary skill in the art to practice or test the parameter widely to find those that are functional or optimal which then would be inclusive or cover the steps as instantly claimed. Absent any teaching of criticality by the Applicant concerning the amount of time for NTG exposure, it would be prima facie obvious that one of ordinary skill in the art would recognize that this limitation is a result effective variable which can be met as a matter of routine optimization.
Conclusion
Claims 1-15 are rejected.
No claims are allowed.
Correspondence Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to NATALIE IANNUZO whose telephone number is (703)756-5559. The examiner can normally be reached Mon - Fri: 8:30-6:00 EST.
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, Sharmila Landau can be reached at (571) 272-0614. 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.
/NATALIE IANNUZO/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
Prosecution Insights