US-12624401-B2 - Single input multiplex decision systems and methods of using the same

Abstract

The present invention is directed to a single input multiplex decision expression system, and a method of using same, such as for expressing a reporter protein.

Inventors

• Luna RIZIK
• Ramez Daniel

Assignees

• TECHNION RESEARCH & DEVELOPMENT FOUNDATION LIMITED

Dates

Publication Date

20260512

Application Date

20200528

Claims (20)

1. 1 . A system comprising: a first expression vector comprising a first promoter sequence operably linked to a sequence encoding a first regulatory protein, wherein said first promoter is responsive to an input signal, and further comprising a second promoter sequence operably linked to a sequence encoding a second regulatory protein, wherein said second promoter is responsive to said first regulatory protein, and wherein said first expression vector is a low copy plasmid; a second expression vector comprising a third promoter sequence operably linked to a sequence encoding a first output protein, wherein said third promoter is responsive to said second regulatory protein and wherein said sequence encoding said first output protein further comprises a degradation tag, and further comprising a first copy of a fourth promoter sequence located between said third promoter and said sequence encoding said first output protein, wherein said first copy of said fourth promoter sequence transcribes in a direction opposite to said third promoter sequence, and further comprising a fifth promoter sequence operably linked to a sequence encoding said first output protein, wherein said fifth promoter is responsive to said first regulatory protein, and wherein said second expression vector is a high copy plasmid; and a third expression vector comprising a second copy of said fourth promoter operably linked to a sequence encoding a second output protein, and wherein said third expression vector is a medium copy plasmid.
2. 2 . The system of claim 1 , wherein: (i) said first promoter is responsive to said first regulatory protein; (ii) said second regulatory protein is expressed upon activation of said second promoter; and (iii) said first output protein of said second expression vector is expressed, upon activation of said fifth promoter.
3. 3 . The system of claim 2 , wherein said first regulatory sequence is located between said third promoter and said sequence encoding said first output protein comprising a degradation tag of said second expression vector, and wherein said second regulatory sequence is located between said fourth promoter and said sequence encoding said second output protein of said third expression vector.
4. 4 . The system of claim 2 , wherein any one of said first and said second regulatory sequences of said system is a non-coding polynucleotide.
5. 5 . The system of claim 4 , wherein said non-coding polynucleotide is a catalytic polynucleotide.
6. 6 . The system of claim 5 , wherein said catalytic polynucleotide is a ribozyme.
7. 7 . The system of claim 1 , wherein (i) said first promoter sequence and said sequence encoding a first regulatory protein of said system, and said second promoter sequence and said sequence encoding a second regulatory protein of said system, are located on separate expression vectors; and (ii) wherein said third promoter sequence and said sequence encoding a first output protein comprising a degradation tag of said system, said fourth promoter sequence, and said fifth promoter sequence and said sequence encoding said first output protein of said system, are located on separate expression vectors.
8. 8 . The system of claim 1 , wherein any one of: (i) said first promoter of said system is a mutated variant promoter of said system (a) or of any one of said first copy and said second copy of said fourth promoter of said system; (ii) wherein said first regulatory protein of said system binds to said mutated variant promoter of said system with reduced binding affinity compared to the binding affinity of said first regulatory protein to any one of said first copy and said second copy of said fourth promoter of said system; (iii) wherein said first promoter of said system is activated when bound to said input signal; (iv) wherein when said first promoter of said system is activated said first regulatory protein of said system is expressed; (v) wherein in said system said second promoter, said first copy and said second copy of said fourth promoter are activated when bound to said first regulatory protein; and (vi) wherein said third promoter said system is activated when bound to said second regulatory protein.
9. 9 . A cell or a cell lysate comprising the system of claim 1 , optionally wherein said cell is a bacterial cell.
10. 10 . The cell or cell lysate of claim 9 , further comprising an input signal.
11. 11 . A composition comprising the cell or cell lysate of claim 9 .
12. 12 . A method for expressing one or more output proteins using a single input signal, the method comprising: a. providing a cell or a cell lysate comprising said system of claim 1 ; b. contacting said cell or said cell lysate with an input signal capable of activating said system; and c. detecting the expression of any one of said first and said second output proteins of said system in said cell or said cell lysate, optionally wherein said contacting comprises contacting said cell or said cell lysate with different concentrations of said input signal.
13. 13 . The method of claim 12 , further comprising a step of determining the expression levels of any one of said first and said second output proteins.
14. 14 . A method for converting an analog signal to a digital output in a cell, the method comprising contacting said cell with the system of claim 1 , thereby converting an analog signal to a digital output in the cell.
15. 15 . The system of claim 1 , wherein said first regulatory protein binds to said fourth promoter with greater affinity than said first regulatory protein binds to said first promoter or said second promoter.
16. 16 . The system of claim 1 , wherein when said fourth promoter is activated said second output protein is expressed, expression of said first output protein comprising a degradation tag from said second expression vector is repressed, or any combination thereof.
17. 17 . The system of claim 1 , wherein when said third promoter is activated said first output protein comprising a degradation tag of said second expression vector is expressed.
18. 18 . The system of claim 1 , wherein said second expression vector of further comprises a first regulatory sequence, and said third expression vector further comprises a second regulatory sequence.
19. 19 . The system of claim 18 , wherein said first regulatory sequence is located between said fourth promoter and said sequence encoding said first output protein comprising a degradation tag of said second expression vector.
20. 20 . The system of claim 1 , wherein said first regulatory sequence comprises a ribosomal binding site.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a National Phase of PCT Patent Application No. PCT/IL2020/050601 having International filing date of May 28, 2020, which claims the benefit of priority of U.S. Provisional Patent Application No. 62/853,285 titled “SINGLE INPUT MULTIPLEX DECISION SYSTEMS AND METHODS OF USING THE SAME”, filed May 28, 2019, and of U.S. Provisional Patent Application No. 62/872,800 titled “SINGLE INPUT MULTIPLEX DECISION SYSTEMS AND METHODS OF USING THE SAME”, filed Jul. 11, 2019, the contents of which are incorporated herein by reference in their entirety. FIELD OF INVENTION The present invention, in some embodiments, thereof, is in the field of molecular biology and genetic engineering. BACKGROUND Many applications of genetic or metabolic engineering require inducible expression of the desired pathway. Inducible promoters are one of the easiest and most effective ways to control gene expression. However, in more sophisticated applications there is a need to coordinate expression of multiple genes. The acceptable/common solution utilizes different inducible promoters for different genes. The downside of this approach is that it requires multiple inducers which can be expensive and/or hard to obtain/produce. Moreover, the use of different inducers may cause crosstalk and/or cell toxicity. SUMMARY The present invention, in some embodiments thereof, is directed to a single input multiplex decision expression system, and methods of use thereof, such as for expressing a reporter protein. According to a first aspect, there is provided a system comprising: (a) a first expression vector comprising a first promoter sequence operably linked to a sequence encoding a first regulatory protein, wherein the first promoter is responsive to an input signal, and further comprising a second promoter sequence operably linked to a sequence encoding a second regulatory protein, wherein the second promoter is responsive to the first regulatory protein; (b) a second expression vector comprising a third promoter operably linked to a sequence encoding a third regulatory protein; and (c) a third expression vector comprising a fourth promoter sequence operably linked to a sequence encoding an output protein, wherein the fourth promoter is responsive to the second regulatory protein, and further comprising a fifth promoter sequence located between the fourth promoter and the sequence encoding the output protein, wherein the fifth promoter is responsive to the first regulatory protein and to the third regulatory protein, and wherein the fifth promoter sequence transcribes in a direction opposite to the fourth 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 sequence encoding a first regulatory protein, wherein the first promoter is responsive to an input signal, and further comprising a second promoter sequence operably linked to a sequence encoding a second regulatory protein, wherein the second promoter is responsive to the first regulatory protein; (b) a second expression vector comprising a third promoter sequence operably linked to a sequence encoding a first output protein, wherein the third promoter is responsive to the second regulatory protein and wherein the sequence encoding the first output protein further comprises a degradation tag, and further comprising a fourth promoter sequence located between the third promoter and the sequence encoding the first output protein, wherein the fourth promoter sequence transcribes in a direction opposite to the third promoter sequence, and further comprising a fifth promoter sequence operably linked to a sequence encoding the first output protein, wherein the fifth promoter is responsive to the first regulatory protein; and (c) a third expression vector comprising the fourth promoter operably linked to a sequence encoding a second output protein. According to another aspect, there is provided a cell or a cell lysate comprising the system of the invention. According to another aspect, there is provided a composition comprising the cell or cell lysate of the invention. According to another aspect, there is provided a method for expressing one or more output proteins using a single input signal, the method comprising: (a) providing a cell or a cell lysate comprising the system of the invention; (b) contacting the cell or the cell lysate with an input signal capable of activating the system; and (c) detecting the expression of the one or more output proteins in the cell or the cell lysate. According to another aspect, there is provided a method for converting an analog signal to a digital output in a cell, the method comprising contacting the cell with the system of the invention, thereby converting an analog signal to a digital output in the cell. In some embodiments, one of: (i) the first promoter is responsive to the first reg