The present invention relates to methods and systems for tracking bioremediation processes. More particularly, the invention relates to methods for the convenient expression of genes of Dehalococcoides and other relevant microorganisms for remediation and bioenergy and the mass spectrometric detection of gene expression products for tracking the progress of bioremediation and bioenergy related efforts during cleanup of chloroethene-contaminanted environments.
The present invention shows an example of how this invention is applied to detect and track a reductive dehalogenase enzyme in Dehalococcoides i.e, TceA, however this over expression and detection system can be applied to track many other enzymes of interest.
Microorganisms of the family Dehalococcoides are known to perform the beneficial biotransformation of toxic chlorinated ethenes to non-toxic ethene at hazardous waste sites. At the community level, Dehalococcoides performance can be improved by the presence of homoacetogens, where the presence of methanogens diverts electrons and slows down dechlorination. Quantification of Dehalococcoides sp. using isolated DNA and quantitative PCR [1-2] has become one of multiple lines of evidence for remediation decision making; however, the information obtained based on these DNA-based assays is limited. The current assays target 3 identified RDases tceA [3], bvcA [4], and vcrA [5]. Two key limitations are: 1. The primers can be too specific and therefore may not fully capture homolog proteins, which serve the same function but are slightly different at the gene level, and 2. Quantifying Dehalococcoides genes provides no information on microbial interactions, and how these interactions affect dechlorination rates.
The present invention overcomes these two limitations. The disclosed methods and systems have the potential to enhance or even replace existing procedures and materials for the determination of bioremediation progress. The technology disclosed herein overcomes existing limitations in the availability and production of proteins for use as bioremediation agents and as diagnostic materials. The instant over-expression method will allow development of more specific and sensitive protein detection methods which are helpful to detect Dehalococcoides enzymes but could also be applied to many other systems.
This summary is provided to introduce, in a simplified form, a selection of concepts that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
A method of over-expressing proteins originating in Dehalococcoides where gene expression products are separated (e.g., on an SDS gel), and where the gene expression products include proteins encoded by a pBAD18 +a gene of interest, such as, for example, a tceAB construct. The protein gels are refrigerated at 4° C. and later used for de novo sequencing of peptides using proteomic mass spectrometry.
In one aspect, the gel comprises a 5-20% SDS-polyacrylamide gel.
In another aspect a mass spectrometric method for tracking the expression of genes of Dehalococcoides includes the steps of acquiring an environmental sample, extracting proteins and peptides contained therein, introducing proteins and peptides originating from said sample into a mass spectrometer, and analyzing the resultant mass spectra by utilizing reference spectra from overexpressed proteins and peptides from Dehalococcoides.
In another aspect the method is modified by the use of labeled proteins and peptides for target quantification.
In another aspect the method is modified by using isotope labeled peptides for (absolute) quantitation of target proteins and peptides.
In another aspect, a mass spectrometric method for tracking the expression of genes of Dehalococcoides includes acquiring an environmental sample, extracting proteins and peptides contained therein, introducing proteins and peptides originating from said sample into a mass spectrometer, and monitoring ions featuring a mass-to-charge ratio (m/z) specific to peptides resulting from protein digestion with a suitable enzyme (e.g., trypsin) of expression products of the target genes of interest (e.g., tceA [3], bvcA [4], and vcrA).
In another aspect a method for expression of engineered constructs containing sequences coding for functional genes of interest in E. coli strains includes fusing tceA and tceB to an inducible, active promoter to optimize transcription of the open reading frames, and an introduced consensus Shine-Dalgarno sequence allowing for optimal ribosome binding and translation of the open reading frames, and codons optimized for expression in E. coil.
In another aspect, a method of utilizing the metabolic capability of a living organism, with the method includes (a) performing codon optimization of the gene sequence to be expressed for the specific expressing host; (b) using a transforming vector; (c) adding a strong promoter and ribosomal binding sequences for increased expression of the protein of interest; and (d) detecting the over expressed protein using mass spectrometric methods.
While the novel features of the invention are set forth with particularity in the appended claims, the invention, both as to organization and content, will be better understood and appreciated, along with other objects and features thereof, from the following detailed description taken in conjunction with the drawings, in which:
In the drawings, identical reference numbers identify similar elements or components. The sizes and relative positions of elements in the drawings are not necessarily drawn to scale. For example, the shapes of various elements and angles are not drawn to scale, and some of these elements are arbitrarily enlarged and positioned to improve drawing legibility. Further, the particular shapes of the elements as drawn, are not intended to convey any information regarding the actual shape of the particular elements, and have been solely selected for ease of recognition in the drawings.
The following disclosure describes tracking bioremediation processes. Several features of methods and systems in accordance with example embodiments are set forth and described in the FIG.s. It will be appreciated that methods and systems in accordance with other example embodiments can include additional procedures or features different than those shown in the FIG.s. Example embodiments are described herein with respect to analysis of environmental conditions. However, it will be understood that these examples are for the purpose of illustrating the principles, and that the invention is not so limited. Additionally, methods and systems in accordance with several example embodiments may not include all of the features shown in the FIG.s.
Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
Reference throughout this specification to “one example” or “an example embodiment,” “one embodiment,” “an embodiment” or combinations and/or variations of these terms means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
Generally, as used herein, the following terms have the following meanings when used within the context of contaminant sample collection in aquatic or saturated sedimentary environments:
A “sample” as used herein refers to material, such as environmental material obtained from a remediation site that is suspected of containing, or known to contain, an analytes, as for example, proteins and peptides.
“EcoRI” is a well known endonuclease enzyme isolated from strains of E. coli, and is part of the restriction modification system used in cloning.
“KpnI” is a well known restriction enzyme.
The term “TOP10 strain” as used herein refers to E. coli competent TOP10 strains as supplied for example, by Invitrogen Corporation, now part of Life Technologies of California, US.
The term “TCE” as used herein refers to trichloroethene.
tceA is the gene that encodes for the enzyme TceA. TceA performs the dechlorination (conversion to less chlorinated compounds) of TCE.
RDase is a/any reductive dehalogenase, these are enzymes that convert chlorinated compounds to less chlorinated products.
Referring now to
In one example, the 1792-bp section containing the two genes of interest tceA and tceB was cloned downstream of the arabinose-inducible promoter in plasmid pBAD18. The pBAD18 cloning map includes an optimal Shine Dalgarno sequence followed by genes tceA and tceB and a transcriptional terminator (15 in
E. coli DNA for pBAD18 Cloning Vector (SEQ. NO. 2):
Referring now to
The above over-expressed proteins were used to develop a sensitive proteomic detection approach. Proteomic assays will make use of isotope-labeled peptides to be custom-synthesized based on the peptide mass fingerprints and ionization properties of digested target RDases. These novel proteomic assays will employ the isotope dilution technique for absolute quantitation of proteins in environmental samples (AQUA). Using the capsid protein of the Norovirus (“cruise ship virus”) as a target, we have demonstrated that the use of electrospray and MALDI tandem mass spectrometry allows for the detection of attomole levels of target protein (1 atto mole=1×10−18 moles) [6].
The invention has been described herein in considerable detail in order to comply with the Patent Statutes and to provide those skilled in the art with the information needed to apply the novel principles of the present invention, and to construct and use such exemplary and specialized components as are required. However, it is to be understood that the invention may be carried out by different equipment, and devices, and that various modifications, both as to the equipment details and operating procedures, may be accomplished without departing from the true spirit and scope of the present invention.
The teachings of the following publications are incorporated herein in their entirety by this reference.
[3] Magnuson, J. K., M. F. Romine, D. R. Burris, and M. T. Kingsley. 2000. Trichloroethene reductive dehalogenase from Dehalococcoides ethenogenes: sequence of tceA and substrate range characterization. Appl. Environ. Microbiol. 66:5141-5147.
[4] Krajmalnik-Brown R., T. Hölscher, I. N. Thomson, F. M. Saunders, K. M. Ritalahti, and F. E. Löffler. 2004 “Genetic Identification of a Putative Vinyl Chloride Reductase in Dehalococcoides sp. Strain BAV1”. Applied and Environmental Microbiology. 70(10): 6347-6351.
[5] Müller, J. A., B. M. Rosner, G. von Abendroth, G. Meshulam-Simon, P. L. McCarty, and A. M. Spormann. 2004. Molecular identification of the catabolic vinyl chloride reductase from Dehalococcoides sp. strain VS and its environmental distribution. Appl. Environ. Microbiol. 70:4880-4888.
[6] Colquhoun, D. R., Schwab, K. J., Cole, R. N., and R. U. Halden. 2006. Detection of Norovirus Capsid Protein in Authentic Standards and in Stool Extract by Matrix-Assisted Laser Desorption Ionization and Nanospray Mass Spectrometry. Appl. Environ. Microbiol. 72(4):2749-2755.
Related technology is disclosed in pending PCT patent application publication WO2011/011683, published on Jan. 27, 2011, and entitled “MICROBIAL CULTURES AND METHODS FOR ANAEROBIC BIOREMEDIATION,” to co-inventors of the present application, of which the entire content is incorporated herein by reference.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/US12/43174 | 6/19/2012 | WO | 00 | 3/20/2014 |
Number | Date | Country | |
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61501020 | Jun 2011 | US |