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 .
DETAILED ACTION
Applicant’s amendment filed on 08 September 2025 has been entered. The amendment to the claims does not comply with the requirements of 37 CFR 1.121(c) because the amendment to the claims fails to indicate that claim 10 has been amended. Amendments to the claims filed on or after July 30, 2003 must comply with 37 CFR 1.121(c) which states:
(c) Claims. Amendments to a claim must be made by rewriting the entire claim with all changes (e.g., additions and deletions) as indicated in this subsection, except when the claim is being canceled. Each amendment document that includes a change to an existing claim, cancellation of an existing claim or addition of a new claim, must include a complete listing of all claims ever presented, including the text of all pending and withdrawn claims, in the application. The claim listing, including the text of the claims, in the amendment document will serve to replace all prior versions of the claims, in the application. In the claim listing, the status of every claim must be indicated after its claim number by using one of the following identifiers in a parenthetical expression: (Original), (Currently amended), (Canceled), (Withdrawn), (Previously presented), (New), and (Not entered).
All claims being currently amended in an amendment paper shall be presented in the claim listing, indicate a status of “currently amended,” and be submitted with markings to indicate the changes that have been made relative to the immediate prior version of the claims. The text of any added subject matter must be shown by underlining the added text. The text of any deleted matter must be shown by strike-through except that double brackets placed before and after the deleted characters may be used to show deletion of five or fewer consecutive characters. The text of any deleted subject matter must be shown by being placed within double brackets if strike-through cannot be easily perceived. Only claims having the status of “currently amended,” or “withdrawn” if also being amended, shall include markings. If a withdrawn claim is currently amended, its status in the claim listing may be identified as “withdrawn—currently amended.”
Claims 1-4, 10, 21-23, and 26-27 are amended, claims 24-25 are canceled, and claims 28-30 are new. Claims 1-10 and 21-23, and 26-30 are pending.
The previous claim objections, 112a written description rejection of claims 26-27, 112a enablement rejection of claims 1-10 and 21-22, 112b rejection of claims 1 and 21, and 112b rejection of claim 4 are withdrawn in light of Applicant’s amendment.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
(Maintained) Claim 23 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method for detecting a heavy metal in a sample comprising contacting the sample with Bacillus endophyticus DS43 or with a fluorescent material isolated from Bacillus endophyticus DS43 that exhibits fluorescence at 360-370 nm and that fluorescence is quenched when exposed to As, Cd, Cr, Pb or Hg, does not reasonably provide enablement for a method for detecting a heavy metal in a sample comprising contacting the sample with any other individual fluorescent material that was isolated from Bacillus endophyticus DS43.
The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
As stated in the MPEP §2164.01(a) “There are many factors to be considered when determining whether there is sufficient evidence to support a determination that a disclosure does not satisfy the enablement requirement and whether any necessary experimentation is “undue”.”
In re Wands, 8 USPQ2d 1400 (1988), provided factors to be considered in determining whether a disclosure meets the enablement requirement of 35 U.S.C. 112 (a) or 35 U.S.C. 112 (pre-AIA ), first paragraph. They are:
The nature of the invention
The state of the prior art
The predictability or lack thereof in the art
The amount of direction or guidance present
The presence or absence of working examples
The breadth of the claims
The quantity of experimentation needed, and
The level of skill in the art
It is noted that all of the Wands factors have been considered with regard to the instant claims, with the most relevant factors discussed below.
The breadth of the claims and the nature of the invention: Claim 23 recites the limitation “contacting the sample with Bacillus endophyticus strain DS43 or a subculture thereof with an endogenous fluorescent material which was isolated from Bacillus endophyticus DS43 or a subculture thereof to form a mixture.” The BRI of the claim includes using any individual fluorescent material which was isolated from Bacillus endophyticus strain DS43. This means that the present invention as claimed can be performed with a mixture of any fluorescent compounds that are produced by Bacillus endophyticus strain DS43, or with any one specific fluorescent compound produced by Bacillus endophyticus DS43.
The absence of working examples and the lack of guidance present: The instant disclosure provides working examples for the presently claimed method comprising contacting Bacillus endophyticus strain DS43, or fluorescent extract mixtures from Bacillus endophyticus strain DS43, with a sample comprising heavy metals and irradiating the mixture with 365nm wavelength UV light to detect quenching in fluorescence by heavy metals, and thereby detecting the heavy metals (Examples 1-4). However, there is no working examples or guidance demonstrating the instant method using wavelengths of light other than those wavelengths close to 365nm wavelength UV light, or demonstrating that any individual fluorescent material isolated from Bacillus endophyticus DS43 and used individually would be useful in the present invention. The examples demonstrate the instant method using the DS43 cells themselves (examples 1-3) or a bulk fluorescent acetone extraction that comprises an unknown number of unidentified fluorescent compounds (example 4). The bulk fluorescent acetone extraction does not provide enablement for the full scope of the instant invention because does not narrow down which particular fluorescent compounds within the bulk extract are actually affected by the quenching abilities of the heavy metals. Thus, the bulk fluorescent acetone extraction of Example 4 only provides support for the use of fluorescent acetone extracted mixtures from Bacillus endophyticus strain DS43 in the instant method, but does not provide support for the use of any individual fluorescent material isolated from Bacillus endophyticus DS43 used by itself. Therefore, one of ordinary skill in the art would be required to consult the knowledge in the art for further guidance in this matter.
The state of the prior art and the level of predictability in the art:
It is known in the art that all cells, including bacterial cells, exhibit autofluorescence due to the ubiquitous presence of fluorescent compounds like aromatic chemical compounds and amino acids, flavins, NAD(P)H, lipofuscins, etc., as evidenced by paragraph 1 of Yang et al. (Detection and Quantification of Bacterial Autofluorescence at the Single-Cell Level by a Laboratory-Built High-Sensitivity Flow Cytometer, Anal. Chem. 2012, 84, 1526−1532). This means that the Bacillus endophyticus DS43 cells and their acetone extractions would necessarily comprise a large variety of fluorescent compounds, and thus all of those compounds would be covered under claims 23-24 and useful in the instant method. As discussed above, the instant disclosure does not provide any guidance or working examples demonstrating the use of any specific fluorescent compound isolated from Bacillus endophyticus DS43 individually to detect heavy metals.
Methods for detecting heavy metals using fluorescence quenching in bacterial cells are well known in the art. One such example is Saeed (previously cited and cited below), who teaches a heavy metal detection method by observing changes in fluorescence intensity of fluorescent protein HriGFP engineered to be expressed in a bacterial culture, specifically E. coli (Saeed Pg. 281 Expression of HriGFP in E. coli and Bacillus megaterium). However, detection methods known in the art are usually referencing specific fluorescent compounds, such as the HriGFP taught by Saeed above, and do not often reference the use of entire bacterial cells or bulk extracts acquired therefrom.
The art recognized the benefits and the challenges of fluorescence quenching methods to detect heavy metal ions in samples, as evidenced by Li et al. (Recent advances in fluorescence probes based on carbon dots for sensing and speciation of heavy metals, Nanophotonics 2021; 10(2): 877–908). Li describes several fluorescent sensor performance characteristics, such as sensitivity, reliability, dynamic range, and reproducibility, rely on selecting an appropriate fluorophore. The key factors that will affect the performance of fluorophore for HMs (heavy metals) detection include fluorescence lifetime, fluorescence quantum yield (QY), Stokes shift, the width and shape of its absorption and emission bands, transmission and emission band maxima, molar absorption coefficients, etc.; the fluorescent material must also be stable with sufficient functional groups to allow for efficient interaction with target HMs, and different fluorescent compounds are taught to be designed separately or combined to detect HMs (Li pg. 878 sec. 2 para. 1). Although Li is primarily drawn to the use of carbon dots and not bulk bacterial extracts, Li’s discussion with respect to the limitations and requirements of fluorescent materials is a general discussion not limited to fluorescent carbon dots, and so those limitations and requirements are applicable to all fluorescent materials used in similar detection methods.
With Li’s teachings in mind, one of ordinary skill in the art would recognize that many compound-specific properties affect the ability of a given fluorescent compound to be useful in a method of detecting heavy metals, and as such one of ordinary skill in the art would recognize that not every fluorescent material is suitable for fluorescence quenching detection of heavy metals. Furthermore, because Li teaches that different compounds can be designed specifically for detection of heavy metals, this also indicates that fluorescence quenching detection of heavy metals is not a universal and predictable effect for any given chemical compound. At the very least, one of ordinary skill in the art would conclude that because there are so many compound-specific factors that affect fluorescence quenching heavy metal detection, it would require a substantial amount of undue experimentation to determine if any individual fluorescent compound produced by and/or isolated from Bacillus endophyticus DS43 cells would be successful if used by itself in the instant method.
Thus, one of ordinary skill in the art would not be able to predictably determine if the instant invention can be practiced with any individual fluorescent material isolated from Bacillus endophyticus DS43 by itself, rather than the Bacillus endophyticus DS43 cells themselves, or bulk acetone extractions from those cells.
All of these factors above amount to a burden of undue experimentation on one of ordinary skill in the art to predictably use the present invention commensurate with the full scope of the claims.
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 22-23 and 26-30 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
(Maintained) Regarding claim 22, the claim recites “wherein the fluorescent material comprises one extracted into acetone after cultivating Bacillus endophyticus strain DS43 on tryptone soya agar (TSA), recovering the Bacillus endophyticus DS43, suspending the Bacillus endophyticus DS43 in acetone, and recovering from the acetone an acetone-extracted fraction containing the fluorescent material”. It is unclear if the “fluorescent material” used in the method of claim 1 is now limited to being the entirety of the “acetone-extracted fraction that contains the fluorescent material” such that the acetone itself is also contacting the sample, or if the “fluorescent material” used in the method of claim 1 is the fluorescent material extract from an acetone fraction but only the fluorescent material itself is contacted with the sample.
(Maintained) Regarding claims 26-27, the claims recite “wherein the fluorescent material is yellow when exposed to UV light having a wavelength of 360 nm”. It is unclear if the fluorescent material is intended to just be a material that has a yellow color visibly, under 360nm wavelength light, or if the fluorescent material is intended to fluoresce with a yellow color (emits yellow photons) when irradiated with UV light at a wavelength of 360nm.
(New) Regarding claim 23, the claim phrase “disrupted Bacillus endophyticus strain DS43” on lines 3-4 is indefinite because it is unclear what about the Bacillus endophyticus strain DS43 is being disrupted (the cells, the strain’s genome, the strain’s environment, etc.).
(New) Regarding claims 23 and 29-30, it is unclear what the metes and bounds of the claim term “disrupted” are. It is unclear what actions must be performed or characteristics must be present in order for Bacillus endophyticus strain DS43 to be considered “disrupted”.
Claims 26-30 are dependent on claim 23 and so are indefinite for the same reasons.
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 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.
(Maintained and extended to new claims) Claims 1-5, 9-10, 21-23 and 26-30 are rejected under 35 U.S.C. 103 as being unpatentable over Saeed et al. (HriGFP Novel Fluorescent Protein: Expression and Applications, Molecular Biotechnology (2020) 62:280–288, published 27 February 2020) in view of Ram et al. (Optimization And Characterization Of Intracellular Orange Fluorescent Pigment From Bacillus Endophyticus AVP-9(Kf527823), (2017) Int J Curr Pharm Res, Vol 9, Issue 5, 67-74) and Valli et al. (IN201941052050, published December 20, 2019).
The broadest reasonable interpretation of the method of claim 1 includes a method for detecting a heavy metal in a sample comprising contacting the sample with a fluorescent material which is isolated from Bacillus endophyticus DS43.
Regarding claims 1-5, 9-10, 21-23, and 30, Saeed teaches a heavy metal detection method by observing changes in fluorescence intensity of fluorescent protein HriGFP engineered to be expressed in a bacterial culture, specifically E. coli (Saeed Pg. 281 Expression of HriGFP in E. coli and Bacillus megaterium). HriGFP fluoresces when irradiated with UV light (Saeed Figure 1). The E. coli culture was mixed with different concentrations of different heavy metals and the fluorescence was detected using a fluorometer. The fluorescence levels of the heavy metal samples mixed with the E. coli culture expressing HriGFP were compared to that of a control E. coli HriGFP culture not exposed to heavy metals (Saeed Pg. 281 Heavy Metals Biosensing Ability Test). The heavy metals used in the method as taught by Saeed were arsenic and mercury (commensurate in scope with claims 5 and 9, respectively, as well as claims 1 and 24) (Saeed Page 281 Heavy Metals Biosensing Ability Test).
Saeed does not teach the use of Bacillus endophyticus in the method, nor an irradiating wavelength of 360-370nm UV light.
Ram teaches a strain of Bacillus endophyticus (strain AVP9) that naturally produces a compound that fluoresces under UV light without the need for genetic engineering of the organism to produce foreign fluorescent proteins (Ram Abstract and Page 68 Results and Discussion paragraph 1). Ram also teaches the culturing of the strain AVP9 in nutrient broth, which is functionally equivalent to tryptone soya agar in the instant claims in that the broth cultivates the Bacillus endophyticus strain (Ram Pg. 67 last paragraph). Ram also teaches the recovery of the Bacillus endophyticus cells from the nutrient broth by way of centrifugation (Ram Pg. 68 first paragraph). Ram teaches the acetone extraction of the fluorescent pigment of Bacillus endophyticus AVP9, and the evaporation of the solvents to concentrate the pigment, and furthermore that the fluorescence is maintained after extraction (commensurate in scope with instant claims 4, 22, and 30) (Ram Pg. 68 first paragraph and Pg. 72 Extraction of Pigment and Figure 12).
However, neither Saeed nor Ram teach 360-370nm wavelengths of UV light for irradiating the fluorescent compound or organism, nor the specific bacterial strain Bacillus endophyticus DS43.
Valli teaches that one extracted pigment (chloroxanthomycin) isolated from Bacillus endophyticus AVP9 has an excitation wavelength which has a peak at 365 nm (commensurate in scope with claims 1, 10, and 25) (Valli Figure 5). It is noted that the excitation peak shown in Fig. 5 of Valli overlaps with the claimed range of irradiating UV light. Valli also teaches that chloroxanthomycin fluoresces yellow-green on different media, with or without iron, i.e. independent of iron concentration (commensurate in scope with claim 21) (Valli [004]).
However, Saeed, Ram, and Valli do not teach the specific bacterial strain Bacillus endophyticus DS43.
The Bacillus endophyticus AVP9 taught by Ram and Valli is 99.26% identical to DS43, see BLAST alignment of the 16s rDNA sequences available in GenBank® (BLAST alignment of KF527823 and KU199806.1, page 3). The percent identity of strains AVP9 and DS43 are so close that one of ordinary skill in the art would not consider the two strains to be significantly different, and would find the two strains obvious over one another. Such a miniscule difference in the 16s rDNA sequences between the two strains would not be expected by one of ordinary skill in the art to sufficiently distinguish the two strains from one another such that the claimed method would only be practicable with the instant strain DS43, and not the prior art strain AVP9. Since the prior art teaches that the fluorescent material of AVP9 possesses many of the same characteristics (both fluoresce when irradiated with 365nm UV light, both are isolated from same bacterial species Bacillus endophyticus, an acetone extraction of the fluorescent pigments still fluoresces when exposed to the same light, etc.) as the fluorescent material of DS43, one of ordinary skill in the art would have considered the fluorescent materials produced by the two strains to be the same or so markedly similar such that they would have the same behavioral characteristics to the point where the two strains and the fluorescent materials they produce would be obvious variants of one another.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before of the effective filing date of the instant invention to use Bacillus endophyticus AVP9 and/or an acetone extraction of the fluorescent materials produced therefrom as taught by Ram and Valli in the method of detecting heavy metals in a sample as taught by Saeed. One of ordinary skill in the art would have been motivated to do so because the prior art already taught a method of detecting heavy metals by observing changes in fluorescence of bacterial biosensors, and also that Bacillus endophyticus strain AVP9 was naturally capable of fluorescence without genetic engineering to force the expression of a foreign fluorescent protein; the fact that Bacillus endophyticus AVP9 was naturally capable of fluorescence means that no genetic engineering and expression of foreign fluorescent proteins by bacteria would be necessary to achieve the desired outcome, and thus one of ordinary skill in the art would recognize that it would be less expensive, less time consuming, and more efficient than producing a bacteria that expresses foreign fluorescent proteins. One of ordinary skill in the art would have had reasonable expectations of success because the engineered fluorescence of the E. coli culture of Saeed and the natural fluorescence of Bacillus endophyticus AVP9 taught by Ram and Valli are functionally identical (i.e. both fluoresce when exposed to UV light and are quenched when exposed to heavy metals), and thus would be expected to behave in the same or similar manner when exposed to heavy metals. One of ordinary skill in the art would have been motivated to incorporate the teachings of Valli because Valli teaches the fluorescent characteristics of the same strain of Bacillus endophyticus as that of Ram (strain AVP9). One of ordinary skill in the art would have had reasonable expectations of success because Valli taught that 365 nm wavelength UV light was sufficient to observe fluorescence in Bacillus endophyticus AVP9 and in the acetone extract of the fluorescent pigment therefrom, and thus the method of detecting heavy metals explained above would be functional if irradiating the sample with 365 nm wavelength UV light.
Regarding claims 26-27, Valli teaches that the extracted pigment chloroxanthomycin (also referred to as “AVSR1” within Valli) isolated from Bacillus endophyticus AVP9 has an UV/Vis absorption and excitation spectrum showing absorption/excitation peaking at 515-525nm light, and broad absorption/excitation around 320-390nm, peaking at 365nm (Valli Figure 5). Valli also teaches that chloroxanthomycin (AVSR1) fluoresces yellow-green (Valli [004]), and confirms that it fluoresces yellow when irradiated with 350nm light (Valli Fig. 4). Examiner submits that since the excitation spectrum shown in Valli Fig. 5 shows the same excitation peak encompassing the wavelengths of 350nm, 360nm, and 365nm showing absorption/excitation at similar levels (~0.1-0.2), and since each of those excitation wavelengths are numerically very close to one another meaning they have very similar energy levels as one another, one of ordinary skill in the art would expect the chloroxanthomycin (AVSR1) to be excited when irradiated with UV light at each of those wavelengths, and then fluoresce slightly different wavelengths of yellow light at each of those irradiating excitation wavelengths.
Regarding claim 28, Saeed teaches exposing their E. coli culture expressing HriGFP to different concentrations of different heavy metals, and detecting the changes in fluorescence using a fluorometer as compared to a control (Saeed Pg. 281 Heavy Metals Biosensing Ability Test); thus Saeed teaches contacting a heavy metal containing sample with cells expressing a fluorescent material. However, Saeed does not teach a Bacillus endophyticus bacteria producing the fluorescent material. Ram teaches a strain of Bacillus endophyticus (strain AVP9) that naturally produces a compound that fluoresces under UV light (Ram Abstract and Page 68 Results and Discussion paragraph 1). Therefore, one of ordinary skill in the art would understand that contacting a heavy metal containing sample with cells of Bacillus endophyticus AVP9 would be functionally identical to contacting the heavy metal containing sample with the fluorescent material produced by the cells of Bacillus endophyticus AVP9. As described above, Bacillus endophyticus AVP9 taught by Ram and Valli is 99.26% identical to DS43, see BLAST alignment of the 16s rDNA sequences available in GenBank® (BLAST alignment of KF527823 and KU199806.1, page 3). The percent identity of strains AVP9 and DS43 are so close that one of ordinary skill in the art would not consider the two strains to be significantly different, and would find the two strains obvious over one another. Therefore, it would have been obvious to one of ordinary skill in the art to contact the heavy metal containing sample of claim 23 with the cells of Bacillus endophyticus AVP9 or DS43.
Regarding claim 29, as discussed in the 112b rejection of claim 29 above, the metes and bounds of the claim term “disrupted” are not clear. For compact prosecution, the term “disrupted” is interpreted to encompass any disruption of any characteristic of Bacillus endophyticus strain DS43, including centrifugation and solvent extraction which would disrupt the Bacillus endophyticus. Ram teaches the recovery of the Bacillus endophyticus cells from the nutrient broth by way of centrifugation (Ram Pg. 68 first paragraph), and the acetone extraction of the fluorescent pigment of Bacillus endophyticus AVP9 (Ram Pg. 68 first paragraph and Pg. 72 Extraction of Pigment and Figure 12).
(Maintained) Claims 6-8 are rejected under 35 U.S.C. 103 as being unpatentable over Saeed in view of Ram and Valli as applied to claims 1-5, 9-10, 22-23, and 26-30 above, and further in view of Bereza-Malcom et al. (Environmental Sensing of Heavy Metals Through Whole Cell Microbial Biosensors: A Synthetic Biology Approach, ACS Synth. Biol. 2015, 4, 535−546.).
Saeed, Ram, and Valli do not teach the detection of heavy metals cadmium, chromium, or lead.
However, Bereza teaches that the heavy metals cadmium, chromium, and lead are toxic heavy metals that are quantified in environmental samples in the same manner as arsenic and mercury (Bereza-Malcom Page 536 first paragraph). It is noted that heavy metals arsenic and mercury were taught in a method to detect heavy metals by Saeed.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art ahead of the effective filing date of the instant invention to try the method of detecting heavy metals explained in the 103 rejection of claim 1 with the heavy metals cadmium, chromium, and lead. One of ordinary skill in the art would have been motivated to do so because heavy metal detection methods were already known in the prior art to be used on the heavy metals arsenic, cadmium, chromium, lead, and mercury, and so one of ordinary skill in the art would have had a reasonable expectation of success in trying to extend the obvious method of claim 1 to detect other heavy metals known in the art to be detectable by the same or similar method.
Response to Arguments
Applicant's arguments and the Rule 132 declaration by Dr. Berekaa filed 08 September 2025 have been fully considered but they are not persuasive.
Regarding Applicant’s argument that claim 23 has been amended to indicate the sources and nature of the fluorescent pigment produced by strain DS43 (Remarks pg. 6 sec. Enablement), the amendment to claim 23 does not limit the fluorescent material to exhibit fluorescence at 360-370nm. Although the wavelength of the UV light used in the method step of irradiating the mixture with UV light is limited to 360-370nm wavelength, this limitation does not limit the fluorescent material to those materials which exhibit fluorescence when irradiated with UV light at 360-370nm.
Regarding Applicant’s argument that claim 22 is clearly written that the fluorescent material is extracted into acetone, which can then be used in the method of claim 1 (Remarks pg. 8 para. 3), it is still unclear if claim 22 limits the “fluorescent material” used in the method of claim 1 to being the entirety of the “acetone-extracted fraction that contains the fluorescent material” such that the acetone within the “acetone-extracted fraction” is also contacting the sample, or if claim 22 limits the “fluorescent material” used in the method of claim 1 to being only the fluorescent material itself which is extractable by acetone but only the fluorescent material itself is contacted with the sample, no acetone.
Regarding Applicant’s argument that the fluorescent material recited in claims 26-27 is yellow under UV illumination as already described in claims 26-27 and thus overcomes the 112b rejection of claims 26-27 (Remarks pg. 8 para. 4), it is still unclear whether the material itself is of yellow color even when under 360nm wavelength UV light, or if the material may not be yellow material but fluoresces yellow colored light when under 360nm wavelength UV light. In other words, the claim makes no indication if the yellow color is just the color of the material and thereby limiting the fluorescent material to be a yellow colored compound, or if the yellow color is the fluorescence color when the material is exposed to 360nm UV light thereby limiting the fluorescent material to fluoresce yellow under 360nm UV light.
Regarding Applicant’s arguments that the pigments of AVP9 and DS43 have significant functional differences as determined by absorption/excitation spectra, specifically that the pigment isolated from AVP9 has an absorption peak of 493nm whereas the pigment isolated from DS43 has an absorption peak of 519nm (Remarks pg. 10 paras. 1 and 3 and Declaration sec 10-25), the fluorescent pigments of AVP9 do have an absorbance peak of around 518nm because Valli teaches that the pigment AVSR1 extracted from AVP9 has an absorption peak at about 515-525nm (Valli Fig. 5) and that chloroxanthomycin (AVSR1) fluoresces yellow-green (Valli [004]), and confirms that it fluoresces yellow when irradiated with 350nm light (Valli Fig. 4). As such, pigment chloroxanthomycin (AVSR1) isolated from AVP9 has a absorption peak of around 518nm.
Regarding Applicant’s arguments that unlike the AVP9 pigment, the DS43 pigment absorbs and emits at peaks about 280, 420, and 519nm, whereas AVP9 exhibits a single peak at 493nm, not multiple peaks (Declaration sec. 15-18), Valli fig. 5 clearly shows that the AVSR1 (chloroxanthomycin) produced and isolated from AVP9 absorbs light at about 280, 420, and 519nm, which matches Applicant’s assertions of DS43.
Regarding Applicant’s arguments that the partial rDNA identity of 99.26% would not indicate that strains AVP9 and DS43 were the same strain or that they would necessarily share genes involved in production of the same or similar fluorescent compounds (Remarks pg. 9 para. 5 and pg. 10 para. 2, and Declaration sections 7-9), the rejection does not state that strains AVP9 and DS43 are the same strain, but rather that the two strains would be obvious over one another in view of not just the miniscule rDNA identity of 99.26%, but also the nearly identical characteristics of the fluorescent materials produced by both strains, namely the identical excitation wavelengths (365nm) and identical capability of fluorescence quenching when exposed to heavy metals. Thus, the genetic and functional similarities of the two strains would suggest to one skilled in the art that the two strains AVP9 and DS43 are obvious over one another.
Regarding Applicant’s arguments that the alleged differences in the structures of the fluorescent pigments of AVP9 and DS43 can be attributed to genetic adaptation of the two strains to their local environments, and furthermore that all Bacillus endophyticus strains necessarily produce fluorescent pigments (Declaration sec. 19-24), there is no evidence presented that shows the small differences in genetic similarity between AVP9 and DS43 have caused any differences in the identity, structures, or characteristics of the fluorescent compounds produced by AVP9 and DS43. Additionally, the discussion of other strains of B. endophyticus which do not produce fluorescent compounds is not particularly relevant at least because those other strains are not claimed and do not have any necessary fluorescent compounds needed to practice the instant invention.
Regarding Applicant’s arguments that the pigments produced by AVP9 are intracellular and thus different from the extracellular pigments produced by DS43 (Remarks pg. 9 para. 4 and Declaration sections 5-6 and 26-29), there is no evidence presented which shows that the pigments produced by AVP9 are intracellular, and thus different from Applicant’s asserted extracellular and intracellular pigments produced by DS43. Any differences in the sources of isolation do not demonstrate any structural differences between the pigments; it only demonstrates that the pigments were isolated differently.
Regarding Applicant’s arguments that the claims refer to a material produced and isolated from strain DS43, not AVP9 (Remarks pg. 12 paras. 2-3), based on the preponderance of evidence, the pigments produced by AVP9 and DS43 appear to be identical or otherwise so close in function so as to be indistinct from one another.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ALEXANDER M DURYEE whose telephone number is (571)272-9377. The examiner can normally be reached Monday - Friday 9:00 am - 5:00 pm.
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/Alexander M Duryee/Examiner, Art Unit 1657
/LOUISE W HUMPHREY/ Supervisory Patent Examiner, Art Unit 1657