Patent Application
20230130375
The present invention is in the field of molecular biology and genetic engineering.
The main challenge of genetic regulatory networks implemented in various synthetic gene circuits in living cells for purpose of sensing, computing, and actuating in fields of diagnostics, health monitoring, environmental monitoring, bioremediation, and metabolic engineering, is the ability to control cellular behavior in modular, robust, and accurate ways.
Untight control of transcriptional regulatory networks applied on promoters, which are the basic transcriptional regulatory component in synthetic circuits, often exhibit high basal level expression or leaky expression of target promoter or part under test (PPUT). In this process, RNAPs bind non-specifically to the target promoter, even in the absence of its inducer molecule, which result in the promoter activity called leakiness. This leakiness also affects the fold change activation (FCA) of the target promoter. FCA is defined as the ratio between the ‘ON’ state, when an activated promoter has maximum activity, and the ‘OFF’ state (background level or basal level), when the promoter has minimum activity. Promoters with high FCA exhibit distinct ON and OFF states in contrast to circuits with low FCA, which are sensitive to environmental changes, have a narrow noise margin and thus, demonstrate poor performance. Tight control of genes involved in metabolic pathways is required to yield high amounts of the desired products in metabolic engineering.
In addition, high basal level expression of enzymes can lead to toxicity to the cells if the expressed protein is toxic. Another aspect of biosensors having high basal level expression is that they often show low sensitivity for detecting target molecule at very low concentrations. To address these problems various biomolecular tools were implemented such as, integrating constitutive expression of a repressor of the target promoter, use a lower copy number plasmid, incorporation of mutations at ribosome binding sites (RBS), addition of degradation tags to decrease protein's half-time, random mutagenesis, hybrid promoter engineering, using a different host cell, antisense transcription, etc.
There is still a great need for a synthetic gene circuit for optimal fold change.
According to a first aspect, there is provided a system comprising at least 2 expression vectors, the system comprising: (a) a first expression vector comprising a first promoter sequence operably linked to a first nucleic acid sequence encoding a gene of interest (GOI) and a second nucleic acid sequence encoding a polynucleotide or a protein product thereof, characterized by being capable of inhibiting or reducing expression of a regulatory RNA polynucleotide, an activity thereof, or both, wherein the first promoter is responsive to an input signal; and (b) a second expression vector comprising a second promoter sequence operably linked to a nucleic acid sequence encoding the regulatory RNA polynucleotide, wherein the second promoter is constitutively active, and wherein the regulatory RNA polynucleotide inhibits or reduces expression levels of the GOI.
According to another aspect, there is provided a system comprising at least 3 expression vectors, the system comprising: (a) a first expression vector comprising a first promoter sequence operably linked to a first nucleic acid sequence encoding a GOI and a second nucleic acid sequence encoding a polynucleotide or a protein product thereof, characterized by being capable of inhibiting or reducing expression of a first regulatory RNA polynucleotide when complexed with a second regulatory RNA polynucleotide, an activity thereof, or both, wherein the first promoter is responsive to an input signal; (b) a second expression vector comprising a second promoter sequence operably linked to a nucleic acid sequence encoding the first regulatory RNA polynucleotide, wherein the second promoter is constitutively active, and wherein the first regulatory RNA polynucleotide inhibits or reduces expression levels of the GOI; and (c) a third expression vector comprising the second promoter sequence operably linked to a nucleic acid sequence encoding the second regulatory RNA polynucleotide.
According to another aspect, there is provided a system comprising an expression vector comprising: (a) a first promoter sequence operably linked to a first nucleic acid sequence encoding a GOI, a second nucleic acid sequence encoding a protein translation regulatory element, and a third nucleic acid sequence encoding a protein characterized by being capable of binding to a second promoter sequence; and (b) a second promoter; wherein the first promoter is responsive to an input signal, wherein the protein characterized by being capable of binding to the second promoter sequence represses transcription from the second promoter, wherein the second promoter is constitutively active, and wherein the second promoter transcribes in a direction opposite to the first promoter sequence.
According to another aspect, there is provided a system comprising: (a) a first expression vector comprising a first promoter sequence operably linked to a first nucleic acid sequence encoding a GOI, a second nucleic acid sequence encoding a transcription factor capable of activating transcription of a second promoter, and a third nucleic acid sequence encoding a first regulatory RNA polynucleotide capable of inhibiting or reducing expression levels of the GOI, the transcription factor, or both, wherein the third nucleic acid sequence is located between the first nucleic acid sequence and the second nucleic acid sequence; and (b) a second expression vector comprising the second promoter sequence operably linked to a nucleic acid sequence encoding a second regulatory RNA polynucleotide, wherein the second promoter is constitutively active, and wherein the second regulatory RNA polynucleotide inhibits or reduces expression of the first regulatory RNA polynucleotide of the first expression vector, an activity thereof, or both.
According to another aspect, there is provided a cell comprising the system disclosed herein.
According to another aspect, there is provided a method for controlling expression level of a GOI operably linked to an inducible promoter in a cell, wherein the promoter is responsive to an input signal, the method comprising contacting the cell of the invention with an effective amount of an agent triggering or providing the input signal, thereby controlling expression level of the GOI operably linked to an inducible promoter in a cell.
According to another aspect, there is provided a method for reducing high basal expression level of a GOI operably linked to an inducible promoter in the absence of an input signal while preserving high expression level of the GOI in the presence of the input signal, wherein the promoter is responsive to the input signal, and wherein the GOI expression is controlled by at least two negative feedback loops, the method comprising contacting a cell comprising the GOI operably linked to the inducible promoter, and having expression being controlled by at least two negative feedback loops, with an effective amount of an agent triggering or providing the input signal, thereby reducing high basal expression level of the GOI operably linked to an inducible promoter in the absence of an input signal while preserving high expression level of the GOI in the presence of the input signal.
According to another aspect, there is provided a method for reducing high basal expression level of a GOI operably linked to an inducible promoter in the absence of an input signal while preserving high expression level of the GOI in the presence of the input signal, wherein the promoter is responsive to the input signal, and wherein the GOI expression is controlled by an indirect coherent feedforward loop, the method comprising contacting a cell comprising the GOI operably linked to the inducible promoter, and having expression being controlled by an indirect coherent feedforward loop, with an effective amount of an agent triggering or providing the input signal, thereby reducing high basal expression level of the GOI operably linked to an inducible promoter in the absence of an input signal while preserving high expression level of the GOI in the presence of the input signal.
In some embodiments, the first expression vector further comprises a third nucleic acid sequence encoding a protein translation regulatory element.
In some embodiments, the third nucleic acid sequence is located between the first nucleic acid sequence and the second nucleic acid sequence.
In some embodiments, the system further comprises a third expression vector comprising the first promoter sequence operably linked to a nucleic acid sequence encoding the polynucleotide or the protein product thereof, characterized by being capable of inhibiting or reducing expression of the regulatory RNA polynucleotide of the second expression vector, an activity thereof, or both.
In some embodiments, the expression vector is devoid of the nucleic acid sequence encoding the polynucleotide or the protein product thereof, characterized by being capable of inhibiting or reducing expression of the regulatory RNA polynucleotide of the second expression vector, an activity thereof, or both.
In some embodiments, the first expression vector further comprises a third nucleic acid sequence encoding a protein translation regulatory element.
In some embodiments, the third nucleic acid sequence is located between the first nucleic acid sequence and the second nucleic acid sequence.
In some embodiments, the system further comprises a fourth expression vector comprising the first promoter sequence operably linked to the nucleic acid sequence encoding the polynucleotide or a protein product thereof, characterized by being capable of inhibiting or reducing expression of the first regulatory RNA polynucleotide when complexed with the second regulatory RNA polynucleotide, an activity thereof, or both.
In some embodiments, the first expression vector is devoid of the nucleic acid sequence encoding a polynucleotide or a protein product thereof, characterized by being capable of inhibiting or reducing expression of the regulatory RNA polynucleotide of the second expression vector, an activity thereof, or both.
In some embodiments, the first expression vector further comprises a fourth nucleic acid sequence encoding a self-cleaving peptide sequence.
In some embodiments, the fourth nucleic acid sequence is located between the third nucleic acid sequence and the second nucleic acid sequence.
In some embodiments, the controlling comprises reducing high basal expression level of the GOI in the absence of the agent, preserving high expression level of the GOI in the presence of the agent, or both.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
All experimental data are averaged from three experiments.
All experimental data represent the average of three experiments.
All experimental data represent the average of three experiments. (8I) A graph showing FCA levels. (8J) A graph showing maximal sensitivity.
The present invention, in some embodiments thereof, is directed to systems comprising a plurality of expression vectors, as well as methods of using same, such as for, controlling expression level of a gene of interest (GOI) operably linked to an inducible promoter in a cell, or reducing high basal expression level of a GOI operably linked to an inducible promoter in the absence of an input signal while preserving high expression level of the GOI in the presence of the input signal.
The present invention, in some embodiments, is applicable in environmental monitoring for detection of toxic chemicals. Improving the FCA together with decreasing basal level expression could be integrated in applications for environmental monitoring for detection of toxic chemical to improve the sensitivity of cell-based biosensors to very low concentrations of target molecules.
The present invention, in some embodiments, is applicable in metabolic engineering, implementation of double negative feedback (DNF) designs into living cells can improve the balance of genes involved in metabolic pathways and ensure a high yield of the target products on the one hand and on the other hand it can downregulate the expression of toxic genes or products.
The present invention, in some embodiments, is applicable in therapeutics, improving the FCA and decreasing the leaky expression may improve safety of genetic circuits for cancer immunotherapy, gene therapy and other biotechnologies.
The present invention, in some embodiments, is applicable in medical diagnosis, for example in the detection of haematuria (a medical term for presence of blood in urine), which can be a serious medical condition. Another example is the detection of glucose in urine, where high glucose can be a sign for a health problem. The implementation of DNF design could improve the biosensing of blood or glucose in urine.
The present invention, in some embodiments, is applicable in cell-free systems (CFS), for improvement of biosensing in portable biosensors, such as biosensors for detection of mercury.
The present invention, in some embodiments, is applicable in cellular immunotherapy. The design could be incorporated within chimeric antigen receptor (CAR) T cell therapy that often shows ON target/OFF tumor toxicities to improve the efficiency and safety of the immunotherapy.
The present invention, in some embodiments, is applicable in virotherapy. The design could be incorporated within the viral genetic load to allow effective activation of transgenes within the specific appropriate target cell. Leakiness in the expression of the transgenes delivered via virotherapy hinders the efficacy of the treatment by either causing expression of genes in unwanted cells or diminishing the difference in expression between correct and incorrect target cells.
According to some embodiments, there is provided a system comprising at least 2 expression vectors, the system comprising: (a) a first expression vector comprising a first promoter sequence operably linked to a first nucleic acid sequence encoding a gene of interest (GOI) and a second nucleic acid sequence encoding a polynucleotide or a protein product thereof, characterized by being capable of inhibiting or reducing expression of a regulatory RNA polynucleotide, an activity thereof, or both, and (b) a second expression vector comprising a second promoter sequence operably linked to a nucleic acid sequence encoding the regulatory RNA polynucleotide.
In some embodiments, the system comprises a plurality of expression vectors. In some embodiments, a plurality comprises or refers to any integer equal to or greater than 2.
In some embodiments, at least 2 comprises: 2-3, 2-4, 2-5, 2-6, 2-7, 3-4, 3-5, 3-6, 4-5, 4-6, or 4-7. Each possibility represents a separate embodiment of the invention.
In some embodiments, the first promoter is responsive to an input signal.
In some embodiments, the second promoter is constitutively active. In some embodiments, the protein product characterized by being capable of inhibiting or reducing expression of a regulatory RNA polynucleotide, attaches to the binding site of the second promoter upstream to the nucleic acid sequence encoding the regulatory RNA polynucleotide, thereby repressing the expression of the nucleic acid sequence encoding the regulatory RNA polynucleotide. In some embodiments, the protein product characterized by being capable of inhibiting or reducing expression of a regulatory RNA polynucleotide, attaches to the binding site of the second promoter upstream to the nucleic acid sequence encoding the regulatory RNA polynucleotide, thereby repressing the expression of the nucleic acid sequence encoding the regulatory RNA polynucleotide and increasing, enhancing, preventing the repression, or any combination thereof, the expression of the GOI.
In some embodiments, the regulatory RNA polynucleotide inhibits or reduces expression levels of the GOI.
In some embodiments, the polynucleotide or a protein product thereof characterized by being capable of inhibiting or reducing expression of a regulatory RNA polynucleotide comprises a decoy protein-binding DNA site. In some embodiments, a decoy protein-binding DNA site comprises a binding-site sponge (BSS). In some embodiments, the polynucleotide or a protein product thereof characterized by being capable of inhibiting or reducing expression of a regulatory RNA polynucleotide comprises a DNA binding protein fused to a repressor. In some embodiments, the DNA binding protein comprises GAL4. In some embodiments, the repressor comprises KRAB. In some embodiments, the polynucleotide or a protein product thereof characterized by being capable of inhibiting or reducing expression of a regulatory RNA polynucleotide comprises GAL4 fused to KRAB (GAL4-KRAB).
In some embodiments, the first expression vector further comprises a third nucleic acid sequence encoding a protein translation regulatory element.
In some embodiments, the protein translation regulatory element increases, enhances, or both, the expression, stability, abundance, or any combination thereof, of the protein product of the GOI. In some embodiments, the protein translation regulatory element comprises or is an internal ribosome entry site (IRES).
In some embodiments, the regulatory RNA polynucleotide comprises or consists of an RNA interfering (RNAi) molecule.
Inhibitory nucleic acids useful in the present methods and compositions include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, modified bases/locked nucleic acids (LNAs), antagomirs, peptide nucleic acids (PNAs), and other oligomeric compounds or oligonucleotide mimetics which hybridize to at least a portion of the target nucleic acid and modulate its function. In some embodiments, the inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof.
As used herein “an interfering RNA” refers to any double stranded or single stranded RNA sequence, capable—either directly or indirectly (i.e., upon conversion)—of inhibiting or down regulating gene expression by mediating RNA interference. Interfering RNA includes but is not limited to small interfering RNA (“siRNA”) and small hairpin RNA (“shRNA”). “RNA interference” refers to the selective degradation of a sequence-compatible messenger RNA transcript.
As used herein “an shRNA” (small hairpin RNA) refers to an RNA molecule comprising an antisense region, a loop portion and a sense region, wherein the sense region has complementary nucleotides that base pair with the antisense region to form a duplex stem. Following post-transcriptional processing, the small hairpin RNA is converted into a small interfering RNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family.
A “small interfering RNA” or “siRNA” as used herein refers to any small RNA molecule capable of inhibiting or down regulating gene expression by mediating RNA interference in a sequence specific manner. The small RNA can be, for example, about 18 to 21 nucleotides long.
As used herein, an “antagomir” refers to a small synthetic RNA having complementarity to a specific microRNA target, with either mispairing at the cleavage site or one or more base modifications to inhibit cleavage. In another embodiment, an “antagomir” refers to a small synthetic RNA having complementarity to a population of microRNA targets, with either mispairing at the cleavage site or one or more base modifications to inhibit cleavage.
In some embodiments, the third nucleic acid sequence is located between the first nucleic acid sequence and the second nucleic acid sequence.
In some embodiments, the system further comprises a third expression vector. In some embodiments, the third expression vector comprises the first promoter sequence operably linked to a nucleic acid sequence encoding the polynucleotide or the protein product thereof, characterized by being capable of inhibiting or reducing expression of the regulatory RNA polynucleotide of the second expression vector, an activity thereof, or both.
In some embodiments, the first expression vector is devoid of the nucleic acid sequence encoding the polynucleotide or the protein product thereof, characterized by being capable of inhibiting or reducing expression of the regulatory RNA polynucleotide of the second expression vector, an activity thereof, or both.
According to some embodiments, there is provided a system comprising at least 3 expression vectors, the system comprising: (a) a first expression vector comprising a first promoter sequence operably linked to a first nucleic acid sequence encoding a GOI and a second nucleic acid sequence encoding a polynucleotide or a protein product thereof, characterized by being capable of inhibiting or reducing expression of a first regulatory RNA polynucleotide when complexed with a second regulatory RNA polynucleotide, an activity thereof, or both; (b) a second expression vector comprising a second promoter sequence operably linked to a nucleic acid sequence encoding the first regulatory RNA polynucleotide; and (c) a third expression vector comprising the second promoter sequence operably linked to a nucleic acid sequence encoding the second regulatory RNA polynucleotide.
In some embodiments, the second promoter is constitutively active. In some embodiments, a constitutive promoter comprises a U6 promoter (PU6).
In some embodiments, the first regulatory RNA polynucleotide inhibits or reduces expression levels of the GOI.
In some embodiments, the polynucleotide or a protein product thereof characterized by being capable of inhibiting or reducing expression of a regulatory RNA polynucleotide comprises a nucleic acid sequence encoding a protein capable of binding to DNA. In some embodiments, a protein capable of binding to DNA comprises an enzyme. In some embodiments, a protein capable of binding to DNA comprises a CRISPR-associated protein (Cas). In some embodiments, the Cas is or comprises Cas9. In some embodiments, the Cas is or comprises a dead-Cas (dCas). In some embodiments, the dCas is or comprises dCas9. In some embodiments, the polynucleotide or a protein product thereof characterized by being capable of inhibiting or reducing expression of a regulatory RNA polynucleotide comprises dCas9 fused to KRAB (dCas9-KRAB).
In some embodiments, the first expression vector further comprises a third nucleic acid sequence encoding a protein translation regulatory element.
In some embodiments, the second regulatory RNA polynucleotide is characterized by being capable of inhibiting or reducing expression of the first regulatory RNA polynucleotide when complexed with the second nucleic acid sequence encoding a polynucleotide or a protein product thereof, characterized by being capable of inhibiting or reducing expression of a first regulatory RNA polynucleotide. In some embodiments, the second regulatory RNA polynucleotide is or comprises a single guide RNA (sgRNA).
In some embodiments, the system further comprises a fourth expression vector comprising the first promoter sequence operably linked to the nucleic acid sequence encoding the polynucleotide or a protein product thereof, characterized by being capable of inhibiting or reducing expression of the first regulatory RNA polynucleotide when complexed with the second regulatory RNA polynucleotide, an activity thereof, or both.
In some embodiments, the first expression vector is devoid of the nucleic acid sequence encoding a polynucleotide or a protein product thereof, characterized by being capable of inhibiting or reducing expression of the regulatory RNA polynucleotide of the second expression vector, an activity thereof, or both.
According to some embodiments, there is provided a system comprising an expression vector comprising: (a) a first promoter sequence operably linked to a first nucleic acid sequence encoding a GOI, a second nucleic acid sequence encoding a protein translation regulatory element, and a third nucleic acid sequence encoding a protein characterized by being capable of binding to a second promoter sequence; and (b) a second promoter.
In some embodiments, the protein characterized by being capable of binding to the second promoter sequence represses transcription from the second promoter.
In some embodiments, the second promoter is constitutively active.
In some embodiments, the second promoter is constitutively active, and transcribes in a direction opposite to the first promoter sequence.
In some embodiments, the second promoter is or comprises a cytomegalovirus promoter (CMV).
According to some embodiments, there is provided a system comprising: (a) a first expression vector comprising a first promoter sequence operably linked to a first nucleic acid sequence encoding a GOI, a second nucleic acid sequence encoding a transcription factor capable of activating transcription of a second promoter, and a third nucleic acid sequence encoding a first regulatory RNA polynucleotide capable of inhibiting or reducing expression levels of the GOI, the transcription factor, or both; and (b) a second expression vector comprising the second promoter sequence operably linked to a nucleic acid sequence encoding a second regulatory RNA polynucleotide.
According to some embodiments, there is provided a system comprising: (a) a first expression vector comprising a first promoter sequence operably linked to a first nucleic acid sequence encoding a first output protein/molecule, and a second promoter; and (b) a second expression vector comprising a third promoter operably linked to a second nucleic acid sequence encoding a first regulatory protein and a third nucleic acid sequence encoding a second output protein/molecule.
According to some embodiments, there is provided a system comprising: (a) a first expression vector comprising a first promoter operably linked to nucleic acid sequence encoding a regulatory protein; and (b) a second expression vector comprising a second promoter operably linked to a nucleic acid encoding an output molecule/protein.
According to some embodiments, there is provided a system comprising: (a) a first expression vector comprising a first promoter sequence operably linked to a first nucleic acid sequence encoding a first output protein/molecule, and a second promoter; (b) a second expression vector comprising a third promoter operably linked to a second nucleic acid sequence encoding a first regulatory protein and a third nucleic acid sequence encoding a second output protein/molecule; and (c) a third expression vector comprising a fourth promoter operably linked to a second regulatory protein.
According to some embodiments, there is provided a system comprising: (a) a first expression vector comprising a first promoter sequence operably linked to a first nucleic acid sequence encoding a first output protein/molecule, and a second promoter; (b) a second expression vector comprising a third promoter operably linked to a second nucleic acid sequence encoding a first regulatory protein; and (c) a third expression vector comprising a fourth promoter operably linked to a second regulatory protein.
In some embodiments, the second regulatory RNA polynucleotide inhibits or reduces expression of the first regulatory RNA polynucleotide of the first expression vector, an activity thereof, or both.
In some embodiments, the first expression vector further comprises a fourth nucleic acid sequence encoding a self-cleaving peptide sequence.
In some embodiments, the fourth nucleic acid sequence is located between the third nucleic acid sequence and the second nucleic acid sequence.
In some embodiments, the second promoter transcribes in a direction opposite to the first promoter sequence.
In some embodiments, the second promoter is located between the first promoter and the nucleic acid sequence encoding the first output protein/molecule.
In some embodiments, the second promoter is located 3′ to the nucleic acid sequence encoding the first output molecule/protein.
In some embodiments, the first expression vector comprises from 5′ to 3′ the first promoter, a nucleic acid sequence encoding the first output molecule, and the second promoter.
For a non-limiting example, first regulatory protein e.g., LuxR protein, as exemplified, modulates the second promoter, e.g., plux promoter, by binding to a region of the plux promoter.
In some embodiments, the second promoter is responsive to the first regulatory protein.
In some embodiments, the first expression vector further comprises at least one “regulatory sequence”. In some embodiments, the first expression vector further comprises a plurality of regulatory sequences. In some embodiments, the regulatory sequence or a plurality thereof regulate transcription of the first output molecule. In one embodiment, the regulatory sequence or plurality thereof regulate translation of an output molecule. In one embodiment, the regulatory sequence or plurality thereof regulate degradation of an output molecule. In some embodiments, the regulatory sequence or plurality thereof comprises a degradation tag. Non-limiting examples of a regulatory sequence include, but are not limited to, a ribosomal binding site (RBS), a riboswitch, a ribozyme, a guide RNA binding site, a microRNA binding site, a cis-repressing RNA, a siRNA binding site, and a protease target site. Regulatory sequences are typically located between a promoter and a nucleic acid to which it is operably linked such that the regulatory sequences is capable of regulating transcription and/or translation of the downstream (3′) nucleic acid and/or output molecule/protein. In some embodiments, a regulatory sequence or plurality thereof is located in the 5′ untranslated region (UTR) of a polynucleotide encoding an output molecule (e.g., gene or a transcript thereof). In some embodiments, a regulatory sequence or plurality thereof is located in the 3′ UTR of a nucleic acid and controls degradation of the nucleic acid. In some embodiments, a regulatory sequence or plurality thereof is transnationally-fused to a protein-coding sequence so as to affect stability and/or intracellular-localization of the protein.
In some embodiments, a regulatory sequence or plurality thereof comprises or is RBS. In some embodiments, the RBS is selected from: RBS30, RBS31, and RBS34.
In some embodiments, a regulatory sequence or plurality thereof comprises or is a ribozyme. In some embodiments is RiboJ.
In some embodiments, the system further comprises a third expression vector comprising the first promoter operably linked to a nucleic acid sequence encoding a second regulatory protein.
In some embodiments, the second promoter is responsive to or is induced by the second regulatory protein. In some embodiments, the second regulatory protein binds to the second promoter. In some embodiments, the second regulatory protein binds to the second promoter thereby inhibiting, blocking, or reducing the expression and/or transcription of the output molecule/protein.
In some embodiments, the third expression vector further comprises the second promoter. In some embodiments, the third expression vector further comprises a regulatory sequence or plurality thereof, as described herein.
In some embodiments, the third expression vector comprises the second promoter being located between the first promoter and the nucleic acid sequence encoding the second regulatory protein.
In some embodiments, the third expression vector comprises the second promoter being located 3′ to the nucleic acid sequence encoding the second regulatory protein.
In some embodiments, the third expression vector comprises from 5′ to 3′ the first promoter, a nucleic acid sequence encoding the second regulatory protein, and the second promoter.
In some embodiments, the second promoter is responsive to or is induced by the regulatory protein. In some embodiments, the regulatory protein binds to the second promoter. In some embodiments, the regulatory protein binds to the second promoter thereby inhibiting, blocking, or reducing the expression and/or transcription of the output molecule/protein.
In some embodiments, the regulatory protein is negatively regulated by a signal molecule, thereby is inhibited. In some embodiments, an inhibited regulatory protein does not inhibit, block, or reduce the expression and/or transcription of the output molecule/protein.
In some embodiments, the system further comprises a fourth expression vector comprising the first promoter operably linked to a third regulatory protein.
In some embodiments, the first regulatory protein modulates or controls the expression derived from or driven or by the second promoter of the first expression vector.
In some embodiments, the second regulatory protein modulates or controls the expression derived from or driven by the first promoter of the first expression vector.
In some embodiments, the third regulatory protein modulates or controls the expression derived from or driven by the fourth promoter of the third expression vector.
The term “promoter” as used herein refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for regulatory proteins, such as a transcriptional activator or repressor.
In some embodiments, the promoter is operably linked to a polynucleotide sequence, such as, but not limited to encoding a gene or polynucleotide of interest (i.e., targeted for expression).
The term “operably linked” is intended to mean that the polynucleotide sequence of interest is linked to the regulatory element or elements in a manner that allows for expression of the polynucleotide sequence (e.g., in an in vitro transcription/translation system, artificial cell, or in a host cell when the expression vector is introduced into the artificial or host cell).
In some embodiments, a promoter is considered “responsive” to an input signal if the input signal modulates the function of the promoter, indirectly or directly. In some embodiments, an input signal may positively modulate a promoter such that the promoter activates or increases, transcription of a nucleic acid to which it is operably linked. In some embodiments, an input signal may negatively modulate a promoter such that the promoter is prevented from activating or inhibits, or decreases, transcription of a nucleic acid to which it is operably linked. In some embodiments, an input signal modulates the function of the promoter directly by binding to the promoter or by acting on the promoter without an intermediate signal.
According to the present invention, in some embodiments thereof, a promoter responsive to an input signal and/or regulatory protein is considered an “inducible” promoter. Inducible promoters for use according to the present invention include any inducible promoter described herein or known to one of ordinary skill in the art. Non-limiting examples of inducible promoters include, but are not limited to, chemically-regulated, biochemically-regulated, and/or physically-regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and/or other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO), and/or a tetracycline trans-activator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature/heat-inducible promoters (e.g., heat shock promoters), and/or light-regulated promoters (e.g., light responsive promoters from plant cells), or any combination thereof.
In some embodiments, the gene of interest may encode any protein which a skilled artisan wishes to produce (hereinafter “a polypeptide of interest”). In some embodiments, the polypeptide of interest is a full protein. In some embodiments, the polypeptide of interests is a fragment of a protein. In some embodiments, the polypeptide of interests is an enzyme. In some embodiments, the polypeptide of interests is an antibody. In some embodiments, the polypeptide of interests is a therapeutic protein. In some embodiments, the polypeptide of interest is a structural protein. In some embodiments, the polypeptide of interest is a scaffold protein. In some embodiments, the polypeptide of interest is a reporter gene. In some embodiments, the polypeptide of interest is a heterologous protein. In some embodiments, the polypeptide of interest is industrially relevant protein. Examples of industrial and pharmaceutically relevant proteins include, but are not limited antibodies, antibody fragments, hormones, interleukins, enzymes, coagulants and vaccines to name but a few. Specific examples of proteins include, but are not limited to, insulin, thyroid hormone, human growth hormone, follicle-stimulating hormone, factor VIII, erythropoietin, granulocyte colony-stimulating factor, alpha-galactosidase A, alpha-L-iduronidase, N-acetylgalactosamine-4-sulfatase, interferon, insulin-like growth factor 1, and lactase.
As used herein, the term “input signal” refers to any chemical (e.g., small molecule) or non-chemical (e.g., light or heat) signal in a cell, or to which the cell is exposed, that modulates, i.e., activates or inhibits, directly or indirectly, a component (e.g., a promoter) of a system as disclosed herein. In some embodiments, an input signal is a biomolecule that modulates the function of a promoter (referred to as direct modulation), or is a signal that modulates a biomolecule, which in turn modulates the function of the promoter (referred to as indirect modulation). In some embodiments, an input signal is endogenous to a cell or a normally exogenous condition, compound, protein, or any combination thereof, that contacts a promoter of a system as disclosed herein in such a way as to be active in modulating (e.g., inducing or repressing) transcriptional activity from a promoter responsive to the input signal (e.g., an inducible promoter).
Non-limiting examples of chemical input signals include, but not limited to, signals extrinsic or intrinsic to a cell, such as amino acids and amino acid analogs, saccharides and polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal containing compounds, salts, ions, enzymes, enzyme substrates, enzyme substrate analogs, hormones, quorum-sensing molecules, and others.
Non-limiting examples of non-chemical input signals include, without limitation, changes in physiological conditions, such as changes in pH, light, temperature, radiation, osmotic pressure, saline gradients, or any combination thereof.
In some embodiments, there is provided a cell comprising a system as disclosed herein.
In some embodiments, the cell is a transgenic cell. In some embodiments, the cell is a transformed cell. In some embodiments, the cell is a transfected cell.
In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a bacterial cell.
In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is an animal cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a mammalian cell.
As used herein, the terms “transgenic”, “transformed”, and “transfected” “cell” refers to any cell that has undergone human manipulation on the genomic or gene level. In some embodiments, the transgenic cell has had exogenous polynucleotide(s) and/or expression vector(s), such as disclosed herein, introduced into it. In some embodiments, a transgenic cell comprises a cell that has an artificial vector introduced into it. In some embodiments, a transgenic cell is a cell which has undergone genome mutation or modification. In some embodiments, a transgenic cell is a cell that has undergone CRISPR genome editing. In some embodiments, a transgenic cell is a cell that has undergone targeted mutation of at least one base pair of its genome. In some embodiments, the exogenous expression vector(s) (e.g., of a system as disclosed herein) is/are stably integrated into the cell. In some embodiments, the transgenic cell expresses a nucleic acid sequence of an expression vector of a system as disclosed herein. In some embodiments, the transgenic cell expresses a vector of the invention. In some embodiments, the transgenic cell expresses a gene of interest, or a polypeptide encoded therefrom.
Expressing of a gene within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell's genome. In some embodiments, the gene is in an expression vector such as plasmid or viral vector. One such example of an expression vector containing p16-Ink4a is the mammalian expression vector pCMV p16 INK4A available from Addgene.
A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.
The vector may be a DNA plasmid delivered via non-viral methods or via viral methods. The viral vector may be a retroviral vector, a herpesviral vector, an adenoviral vector, an adeno-associated viral vector or a poxviral vector. The promoters may be active in mammalian cells. The promoters may be a viral promoter.
In some embodiments, the vector is introduced into the cell by standard methods including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)),Heat shock, infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327. 70-73 (1987)), and/or the like.
In some embodiments, nucleic acid sequences are transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA.
In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205. Other exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
In some embodiments, recombinant viral vectors, which offer advantages such as lateral infection and targeting specificity, are used for in vivo expression. In one embodiment, lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.
In one embodiment, plant expression vectors are used. In one embodiment, the expression of a polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)] are used. In another embodiment, plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3:1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)] or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.
It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.
A person with skill in the art will appreciate that a gene can also be expressed from a nucleic acid construct administered to the individual employing any suitable mode of administration, described hereinabove (i.e., in vivo gene therapy). In one embodiment, the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the individual (i.e., ex vivo gene therapy).
The term “expression profile” refers to expression of a group/set of genes. In some embodiments, the expression profile may be detected at the expression levels such as by analyzing and determining RNA values (e.g., mRNA or miRNA). The RNA levels may be determined in various samples including but not limited to cells, and exosomes. In some embodiments, the expression profile may be detected at the translation levels such as by analyzing and determining CTAs expressed on a cell surface such as by using antibodies.
As used in reference with the methods of the invention, “increase in expression of the expression profile” refers to a sum increase of expression of the specific set of CTAs. For a non-limiting example, a specific value of increase may be a result of increase of all the antigens of the set. Alternatively, specific value of increase may be a result of increase of only a few antigens of the set. In some embodiments, the increase refers to at least 10% increase, 20% increase, 30% increase, 40% increase, 50% increase, 60% increase, 70% increase, 80% increase, 90% increase, 100% increase in expression level of the expression profile.
A variety of known techniques may be suitable for determining an expression profile. Such techniques include methods based on hybridization analysis of polynucleotides and on sequencing of polynucleotides, and proteomics-based methods. In some embodiments, the determining step is performed by nucleic acid hybridization, nucleic acid amplification, or an immunological method. In some embodiments, the determining step is performed in-situ. In some embodiments, fluorescence labeling or staining are applied. In some embodiments, an imaging step is further applied.
In some embodiments, the expression profile is obtained by measuring protein levels of CT antigens. In some embodiments, the expression, and the level of expression, of proteins or polypeptides of interest can be detected through immunohistochemical staining of tissue slices or sections. Additionally, proteins/polypeptides of interest may be detected by Western blotting, ELISA or Radioimmunoassay (MA) assays employing protein-specific antibodies.
Alternatively, protein levels can be determined by constructing an antibody microarray in which binding sites comprise immobilized, preferably monoclonal, antibodies specific to a plurality of proteins of interest. Methods for making monoclonal antibodies are well known (see, e.g., Harlow and Lane, 1988, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor, N.Y., which is incorporated in its entirety for all purposes). In one embodiment, monoclonal antibodies are raised against synthetic peptide fragments designed based on genomic sequence of the cell. With such an antibody array, proteins from the cell are contacted to the array, and their binding is assayed with assays known in the art.
In some embodiments, the determining step comprises the step of obtaining nucleic acid molecules from a biological sample. In some embodiments, the nucleic acids molecules are selected from mRNA molecules, DNA molecules and cDNA molecules. In some embodiments, the cDNA molecules are obtained by reverse transcribing the mRNA molecules. In some embodiments, the expression profile is determined by measuring mRNA levels of CT antigens. Methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and Locker, Lab Invest. 56:A67 (1987), and De Andres et al., BioTechniques 18:42044 (1995).
Numerous methods are known in the art for measuring expression levels of a one or more gene such as by amplification of nucleic acids (e.g., PCR, isothermal methods, rolling circle methods, etc.) or by quantitative in situ hybridization. Design of primers for amplification of specific genes is well known in the art, and such primers can be found or designed on various websites.
The skilled artisan will understand that these methods may be used alone or combined. Non-limiting exemplary method are described herein.
RT-qPCR: A common technology used for measuring RNA abundance is RT-qPCR where reverse transcription (RT) is followed by real-time quantitative PCR (qPCR). Reverse transcription first generates a DNA template from the RNA. This single-stranded template is called cDNA. The cDNA template is then amplified in the quantitative step, during which the fluorescence emitted by labeled hybridization probes or intercalating dyes changes as the DNA amplification process progresses. Quantitative PCR produces a measurement of an increase or decrease in copies of the original RNA and has been used to attempt to define changes of gene expression in cancer tissue as compared to comparable healthy tissues.
RNA-Seq: RNA-Seq uses recently developed deep-sequencing technologies. In general, a population of RNA (total or fractionated, such as poly(A)+) is converted to a library of cDNA fragments with adaptors attached to one or both ends. Each molecule, with or without amplification, is then sequenced in a high-throughput manner to obtain short sequences from one end (single-end sequencing) or both ends (pair-end sequencing). The reads are typically 30-400 bp, depending on the DNA-sequencing technology used. In principle, any high-throughput sequencing technology can be used for RNA-Seq. Following sequencing, the resulting reads are either aligned to a reference genome or reference transcripts or assembled de novo without the genomic sequence to produce a genome-scale transcription map that consists of both the transcriptional structure and/or level of expression for each gene. To avoid artifacts and biases generated by reverse transcription direct RNA sequencing can also be applied.
Microarray: Expression levels of a gene may be assessed using the microarray technique. In this method, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are arrayed on a substrate. The arrayed sequences are then contacted under conditions suitable for specific hybridization with detectably labeled cDNA generated from RNA of a test sample. As in the RT-PCR method, the source of RNA typically is total RNA isolated from a tumor sample, and optionally from normal tissue of the same patient as an internal control or cell lines. RNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g., formalin-fixed) tissue samples. For archived, formalin-fixed tissue cDNA-mediated annealing, selection, extension, and ligation, DASL-Illumina method may be used. For a non-limiting example, PCR amplified cDNAs to be assayed are applied to a substrate in a dense array. Microarray analysis can be performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GenChip technology, or Incyte's microarray technology.
According to some embodiments, there is provided a method for controlling expression level of a gene of interest (GOI) operably linked to an inducible promoter in a cell.
According to some embodiments, there is provided a method for reducing high basal expression level of a GOI operably linked to an inducible promoter in the absence of an input signal while preserving high expression level of the GOI in the presence of the input signal.
In some embodiments, the promoter is responsive to an input signal. In some embodiments, the GOI expression is controlled by at least two negative feedback loops.
In some embodiments, the GOI expression is controlled by an indirect coherent feedforward loop.
In some embodiments, the method comprises contacting a cell as disclosed herein.
In some embodiments, the method comprises contacting a cell comprising a system as disclosed herein.
In some embodiments, the method comprises controlling the expression of GOI, an output molecule/protein or a plurality thereof, or any combination thereof, in a transgenic, transfected, or transformed cell, as disclosed herein, wherein the cell comprises the system disclosed herein.
In some embodiments, the method comprises contacting the cell with an effective amount of an agent triggering or providing an input signal, thereby controlling expression level of the GOI operably linked to an inducible promoter in a cell.
In some embodiments, the method comprising contacting a cell comprising the GOI operably linked to the inducible promoter, and having expression being controlled by at least two negative feedback loops, with an effective amount of an agent triggering or providing the input signal, thereby reducing high basal expression level of the GOI operably linked to an inducible promoter in the absence of an input signal while preserving high expression level of the GOI in the presence of the input signal.
In some embodiments, contacting comprises supplementing the cell with an effective amount of an agent triggering or providing the input signal.
In some embodiments, contacting comprises culturing the cell with or in the presence of an effective amount of an agent triggering or providing the input signal.
In some embodiments, controlling comprises reducing high basal expression level of the GOI in the absence of the agent, preserving high expression level of the GOI in the presence of the agent, or both.
In some embodiments, reducing comprises at least 5%, at least 15%, at least 25%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, at least 97%, at least 99%, or 100% reducing, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, reducing comprises 5-50%, 10-90%, 20-99%, or 5-100% reducing. Each possibility represents a separate embodiment of the invention.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
In the discussion unless otherwise stated, adjectives such as “substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of, or any combination of items it conjoins.
In the description and claims of the present application, each of the verbs, “comprise”, “include”, and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.
Other terms as used herein are meant to be defined by their well-known meanings in the art.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
As used herein, the term “about” when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1,000 nanometers (nm) refers to a length of 1,000 nm±100 nm.
It is noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a polynucleotide” includes a plurality of such polynucleotides and reference to “the polypeptide” includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation.
As used herein, the term “plurality” refers to any integer equal to or greater than 2.
In those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B”.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, chemical and cell biology techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds.) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes Cellis, J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique” by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocols in Immunology” Volumes Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.
Nalidixic acid (NA), hydrogen peroxide (H2O2), sodium (meta)arsenite (AsNaO2), hemin, arabinose, isopropyl-β-D-1-thiogalactopyranoside (IPTG), anhydrotetracyclin hydrochloride (aTc), and acyl homoserine lactone 3OC6HSL (AHL) were used as inducers and were obtained from Sigma-Aldrich.
Standard molecular cloning techniques were used for plasmids construction. New England Biolab's (Beverly, Mass.) restriction endonucleases, Thermo Scientific FastDigest Restriction Enzymes, T4 DNA Ligase were used for plasmid construction. All plasmids in this work were built and transformed to NEB 10-beta Escherichia coli (araD139 D(ara-leu)7697 fhuA lacX74 galK (W80 D(lacZ)M15) mcrA galU recA1 endA1 nupG rpsL (StrR) D(mrr-hsdRMS-mcrBC). The PkatG, PrecA, and ParsR promoters were obtained by PCR amplification (Phusion High-Fidelity PCR Kit—New England Biolabs) from the genome of MG1655 E. coli (F—λ—ilvG—rfb-50 rph-1), with primers listed in Supplementary Information, Table S3 of Litovco et al., 2021 (Nucleic Acids Research, Volume 49, Issue 9, Pages 5393-5406). For part amplification from the genome, 5 ml of MG1655 strain E. coli were inoculated from frozen glycerol stocks and were grown for 16 hours. The next morning, 5 μl from the overgrown culture was mixed with 15 μl of DNase and RNase free water, heated at 96° C. for 6 min and incubated at −80° C. for 10 min. Two (2) μl from this solution was added into PCR mixture with total volume of 50 μl. The primers were synthesized by Integrated DNA Technologies (Leuven, Belgium). Plasmids for cloning were transformed into chemically competent E. coli 10-beta with a standard heat shock protocol. Bacterial cultures were consistently cultured at 37° C. in Luria-Bertani (LB) Broth, Miller (Difco). The overnight grown cells were grown from glycerol stocks in 5 ml at 37° C. or inoculated from colonies on agar plate with appropriate antibiotics for plasmid preparation in the next morning. The growth media was supplemented with appropriate concentration of antibiotics: carbenicillin (50 μg/ml), kanamycin (30 μg/ml), or/and chloramphenicol (34 μg/ml). Plasmids were extracted from the bacterial cells with QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) according to the manufacturer's manual. Colony screening was carried out by PCR with suitable forward and reverse primers. Sequencing was approved by Macrogen Sequencing Service (Macrogen Europe, the Netherlands). All synthetic parts used in this work are listed in Supplementary Information, Table S2 of Litovco et al., 2021 and the plasmid maps are included in Supplementary Information sections 5 and 6 of Litovco et al., 2021.
All plasmids in this work were constructed in a similar manner: Promoter-RB 5-gene-terminator-origin-of-replication-antibiotic-resistance, where the origin-of-replication was changed with AvrII and Sad restriction enzymes, the gene was replaced with restriction enzymes Kpnl and BamHI, and the antibiotic resistance was replaced with SacI and AatII restriction enzymes. Different combinations of plasmids forming different synthetic circuits (are summarized in Supplementary Information, Table S1 of Litovco et al., 2021) were transformed into competent E. coli 10-beta or MG1655 . coli either through heat-shock protocol or electroporation protocol.
Different combinations of plasmids forming different synthetic circuits were transformed into competent NEB 10-beta E. coli for cytometry measurements except from PLhrtO and PrecA, which were transformed into MG1655 E. coli wild type strain. The bacterial cultures were inoculated from colonies on agar plate the previous day and grown in 5 ml of LB with appropriate antibiotics at 37° C. and 300 r.p.m. In the morning, the overnight grown bacterial cultures were diluted 1:100 into fresh LB medium (for PlacO, PLhrtO, and ParsR circuits) or were diluted 1:50 into fresh M9 minimal media (1× M9 Salts (Sigma-Aldrich, M6030), 2 mM MgSO4, 100 μM CaCl2, 0.4% glucose, 0.1% casamino acids, 50 mg/1 thiamine) for the cytometry experiment with appropriate concentration of antibiotics (for PBAD, for PBADsyn, PkatG and PrecA) and incubated for specific time for regrowth and adaptation in fresh media, as described in section 3 for each promoter. Bacterial cultures were transformed into 96-well plates with known concentrations of inducers to total volume of 200 μl, incubated in microplate shaker (37° C., 500 r.p.m) for relevant time described in section 3 for each promoter until they reached optical density OD600 nm ˜0.4-0.7. Then, the fluorescence and scattering of bacterial cultures were analysed through flow cytometry analyzer (CytoFLEX S Flow Cytometer). In all experiments 10,000 events have been obtained and the fluorescence and forward and side scattering were taken using CytExpert 2.2 software. The fluorescence distribution data over population data were extracted together with its geometric mean from each well in 96-well plate and plotted using MATLAB. Fluorescence measurement was based on geometric mean of flow cytometry populations from three experiments. The flow cytometry data for one representative experiment for each combination, which was independently repeated for two more times, is provided in Supplementary Information, section 4 of Litovco et al., 2021. Next, the figures were built in EXCEL, based on geometric mean of flow cytometry populations with error bars representing the standard deviation errors of the geometric mean.
The inventors started the current study by searching for natural biological systems that contain ICF and DNF designs. The inventors found that the ICF network naturally occurs in the L-arabinose utilization system (
The inventors started to implement the ICF and DNF designs in living cells by mimicking a subtraction using a transcriptional interference system and an antisense transcription system. In both systems, the inventors placed the Plux promoter in opposite orientation to promoter under test (PPUT), which inhibits the PPUT activity. The inventors started with the transcriptional interference system (See
According to the herein disclosed simulation results (
As an application the inventors used ICF and DNF designs to improve the performance of different types of bacterial biosensors, specifically for detection of heme, arsenic, hydrogen peroxide, and Nalidixic Acid toxins. Heme is released from lysed red blood cells, and the presence of this biomolecule in clinical samples is indicative of bleeding. The heme biosensor consists of three synthetic parts (
Arsenic is a heavy metal, which can contaminate drinking water and its long-term exposure can lead to toxicity and health issues including skin diseases and cancer. The Arsenic biosensor has an ArsR repressor and a synthetic promoter (ParsR). In the wild-type circuit (
So far, the inventors have applied the proposed designs (OL, ICF and DNF) in biological systems where chemical inputs directly interact with target promoters by binding transcription factors. However, biological systems often activate multiple pathways in response to chemical signals and more specifically in response to toxic chemicals. For example, cells induce repair systems by activating cascades of regulators, e.g. oxidative stress response and SOS response. Implementing the ICF and DNF designs in these complex biological systems can be challenging from a circuit design aspect. To further explore the applicability of the herein disclosed designs, the inventors first examined the oxidative stress response that is sensitive to hydrogen peroxide (H2O2). The transcriptional regulator, OxyR, is activated by oxidation of H2O2 which in turn activates several genes involved in bacterial defense mechanisms, among them is the KatG gene. In the wild-type circuit, the transcriptional activator OxyR binds the katG promoter (PkatG) allowing its activation with FCA level of 10 (
The SOS response in cells is induced by the DNA damage repair process and involves the activation of more than 40 proteins, including recA gene and its transcriptional repressor LexA. To build bacterial biosensors that are sensitive to SOS response, first, a synthetic circuit that includes recA promoter (PrecA) and LexA repressor was integrated into bacterial cells. The circuit is induced by Nalidixic Acid toxin and demonstrated a FCA level of 50 (
The inventors have constructed 7 distinct genetic circuits with the goal of lowering the basal level expression and keeping high expression of a target gene, resulting in an improvement in the fold change activation of the gene downstream to the promoter under test (PPUT).
In the first genetic circuit (
In the second genetic circuit (
In the third genetic circuit (
In the fourth genetic circuit (
In the fifth genetic circuit (
In the sixth genetic circuit (
In the seventh genetic circuit (
While certain features of the invention have been described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.
This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/272,389, titled “TOPOLOGIES OF SYNTHETIC GENE CIRCUIT FOR OPTIMAL FOLD CHANGE ACTIVATION”, filed 27 Oct. 2021, the contents of which are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63272389 | Oct 2021 | US |
