Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1998-02-06
2001-09-11
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C235S2010FS
Reexamination Certificate
active
06287844
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to controlling genetically engineered organisms in the open environment, and in particular, the containment of microorganisms, including but not limited to microorganisms that degrade organic compounds such as aromatic hydrocarbons.
BACKGROUND
Genetically engineered organisms are useful in a variety of settings. Genetically engineered plants offer more efficient sources of food and fuel. Genetically engineered microorganisms (GEMs) offer unlimited supplies of medically useful proteins and also are of interest in the field of bioremediation.
Bioremediation involves the breakdown of toxic compounds by microorganisms and/or their products. Bioremediation is considerably more attractive than merely transporting wastes, as it offers the possibility of degrading toxic compounds to harmless reaction products.
Bioremediation field trials have involved both in-situ and ex-situ treatment methods. Typically, ex-situ treatment involves the transfer of contaminated waste from the site into a treatment tank designed to support microbial growth, i.e., a “bioreactor”. The reactor provides for effective mixing of nutrients and control over temperature, pH and aeration to allow optimum microbial growth.
In-situ treatment involves adding biologicals directly to the waste. This avoids the problems associated with handling (e.g., pumping) toxic compounds. However, in-situ treatment has its own problems. Unlike bioreactors, where microbial growth can be monitored and adjusted, in-situ environmental conditions are difficult to measure and control.
Indeed, it is generally difficult to predict the behavior of genetically engineered organisms in natural ecosystems. There is a concern about the uncontrolled spread of recombinant DNA, including but not limited to the spread of recombinant DNA among indigenous bacterial populations. Potential risk associated with deliberate or unintentional release of GEMs into the open environment can be minimized by the use of debilitated strains. An alternative, and perhaps more appropriate approach is the introduction of conditional or stochastic maintenance functions into GEMs (Molin (1993) Curr. Opin. Biotechnol. 4:299-305; Molin et al. (1993) Annu. Rev. Microbiol. 47:139-166; Ramos et al. (1995) Bio/Technology 13:35-371-3). In such a case, the viability of GEMs depends on the expression of an essential gene or on the repression of a lethal gene controlled by a regulatory promoter responding to changes in the chemical or physical constitution of the environment, or by a promoter undergoing recombinational switches. However, the effectiveness of suicide systems is limited by relatively high frequency of their mutational inactivation, resulting in positive selection of uncontained clones.
Thus, there is a need for better control mechanisms. Such improved approaches should provide better regulation of recombinant gene expression and permit control over the spread of recombinant DNA.
SUMMARY OF THE INVENTION
This invention relates to controlling genetically engineered organisms in the open environrnent, and in particular, the containment of microorganisms, including but not limited to microorganisms that degrade organic compounds such as aromatic hydrocarbons. The present invention contemplates new killing genes and improved strategies to control their expression. The present invention offers a universal conditional lethal system based on the tightly regulated derepression of the streptavidin gene (stv) (Argara-a et al. (1986) Nucleic Acids Res. 14:1871-1882) from the actinobacterium
Streptomyces avidinii
. It targets the metabolism of one-carbon units at the oxidation level of carbon dioxide by depleting an essential prosthetic group, D-biotin (vitamin H); the invention can be used to complement cell suicide systems for which direct targets are cell membranes and walls, or nucleic acids.
In a preferred embodiment, any incompletely repressed expression of the stv gene is eliminated at the level of its transcription, targeting directly the RNA polymerase, as well as at the level of its translation by antisense mRNA. This novel regulatory strategy for containment of GEMs is found to have excellent performance.
It is not intended that the present invention be limited by the specific recombinant organism to be controlled. Indeed, it is not intended that the present invention be limited to hydrocarbon-degrading bacterium. A variety of bacterial and non-bacterial recombinant organisms can be controlled in this manner.
In one embodiment, the present invention contemplates a microorganism comprising a streptavidin gene under control of a promoter, said promoter being negatively regulated by a repressor protein, said repressor protein synthesized in response to an environmental signal (such as the presence of compounds to be degraded). In another embodiment, the present invention contemplates a microorganism comprising a streptavidin gene under control of a first heterologous promoter, said streptavidin gene capable of being transcribed by a heterologous RNA polymerase, said RNA polymerase expressed from a RNA polymerase gene under control of a second heterologous promoter, said second heterologous promoter inhibited by the expression of a repressor protein from a repressor gene, said repressor gene under the control of a third heterologous promoter, said repressor protein expressed by said microorganism in response to an environmental signal (such as the presence of compounds to be degraded). In yet another embodiment, the present invention contemplates a microorganism comprising a streptavidin gene under control of a first heterologous promoter, said streptavidin gene capable of being transcribed by a heterologous RNA polymerase, said RNA polymerase expressed from a RNA polymerase gene under control of a second heterologous promoter, said second heterologous promoter inhibited by the expression of a repressor protein from a repressor gene, said repressor gene under the control of a third heterologous promoter and operably linked to nucleic acid capable (upon transcription) of producing antisense RNA complementary to at least a portion of said streptavidin gene, said first heterologous promoter, or both , said repressor protein expressed by said microorganism in response to an environmental signal (such as the presence of compounds to be degraded).
It is not intended that the present invention be limited to particular polymerases or promoters. In one embodiment, the bacteriophage T7 RNA polymerase is used and the microorganism expresses the polymerase inhibitor, T7 lysozyme.
It is also not intended that the present invention be limited to a particular repressor protein. In one embodiment, the repressor is the
Escherichia coli
LacI repressor.
The present invention also contemplates methods of recombinantly producing the above-described microorganisms by linked the above-described elements in operable combination using recombinant means, as well as compositions comprising the constructs generated by said recombinant means. Finally, the present invention contemplates methods wherein the above-described microorganisms are exposed to the appropriate environment (e.g. a waste source) to cause expression of said repressor protein.
DEFINITIONS
To facilitate understanding of the invention, a number of terms are defined below.
The term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or precursor thereof. The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity is retained.
The term “wild-type” refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene. In contrast, the term “modified” or “mutant” refers to a gene or gene product which displays modificatio
Cantor Charles R.
Kaplan David L.
Mello Charlene
Sano Takeshi
Smith Cassandra L.
Nixon & Peabody LLP
The Trustees of Boston University
Yucel Remy
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