Synthetic transcriptional control elements and methods of generating and using such elements

• US 9,593,338 B2
• Filed: 09/27/2012
• Issued: 03/14/2017
• Est. Priority Date: 09/28/2011
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Liu, Chang, Arkin, Adam P., Qi, Lei S.
• Primary Examiner(s): Leith, Nancy J
• Application Number:
  US14/346,263
• Publication Number:
  US 20150011007A1
• Time in Patent Office: 1,629 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/72 Expression systems using regulatory sequences derived from the lac-operon
• CPC Class Codes: C12N 2830/001 controllable enhancer/promoter combination
• CPC Class Codes: C12N 2840/002 controllable or inducible

Abstract

Provided herein are nucleic acid constructs that contain a synthetic control element that includes a cis-regulator of translation, and an adapter translation-coupled regulator of transcription. Further provided herein are nucleic acid constructs that contain nucleic acid sequences under the control of the synthetic control elements. Also provided are compositions and methods related to the nucleic acid constructs.

Claims

1. A nucleic acid construct comprising a synthetic translation-coupled regulator of transcription (TCRT) comprising in a 5′

to 3′

order;

(a) a cis-regulator of translation that is a RNA-IN variant having a nucleotide sequence selected from the group consisting of SEQ ID NOs;

24-28; and

(b) an adapter TCRT,

wherein (a) and (b) are operably linked so that the transcription of sequences that are 3′

of and operably linked to the synthetic TCRT is regulated by (a).

2. The nucleic acid construct of claim 1, further comprising a nucleic acid sequence of interest that is positioned 3′

of the synthetic TCRT and is operably linked to the synthetic TCRT such that the synthetic TCRT regulates transcription of the nucleic acid sequence of interest.

3. The nucleic acid construct of claim 2, wherein the nucleic acid sequence of interest comprises a sequence selected from the group consisting of:

an insertion site, a sequence encoding a polypeptide, a sequence encoding a sense RNA, a sequence encoding an antisense RNA, a sequence encoding a ribonucleic acid aptamer responsive to an environmental cue, and a sequence encoding an RNA that is responsive to an intracellular or environmental cue.

4. The nucleic acid construct of claim 3, wherein the environmental cue is selected from the group consisting of:

a nucleic acid, a protein, a metabolite, pH, temperature, and combinations thereof.

5. The nucleic acid construct of claim 1, wherein the adapter translation-coupled regulator of transcription comprises in a 5′

to 3′

order;

(a) a polynucleotide encoding the peptide tnaC;

(b) a stop codon; and

(c) a Rho utilization site (rut).

6. The nucleic acid construct of claim 1, wherein the synthetic translation-coupled regulator of transcription (TCRT) has the formula:

(AB)x,

wherein;

(i) A is the cis-regulator of translation;

(ii) B is the adapter TCRT;

(iii) x is the number of repeated AB units;

(iv) x is any integer;

(v) each instance of A can be the same or different cis-regulator of translation as any previous instance of A; and

(vi) each instance of B can be the same or different adapter TCRT as any previous instance of B.

7. A recombinant prokaryotic cell comprising the nucleic acid construct of claim 1.

8. The recombinant prokaryotic cell of claim 7, wherein the cell comprises two or more of the nucleic acid constructs of claim 1, wherein at least two of the cis-regulators of translation are orthogonal.

9. The recombinant prokaryotic cell of claim 8, wherein all of the cis-regulators of translation are orthogonal.

10. The recombinant prokaryotic cell of claim 7, wherein the cell further comprises a polynucleotide that encodes a nucleic acid that binds to the cis-regulator of translation of said nucleic acid construct.

11. The recombinant prokaryotic cell of claim 10, wherein the nucleic acid that binds to the cis-regulator of translation is a taRNA.

12. The recombinant prokaryotic cell of claim 11, wherein the taRNA is taR12 having a sequence of SEQ ID NO:

29.

13. The recombinant prokaryotic cell of claim 10, wherein the nucleic acid that binds to the cis-regulator of translation is an RNA-OUT having the sequence of SEQ ID NO:

30 or an RNA-OUT variant having a sequence selected from the group consisting of;

SEQ ID NO;

31, SEQ ID NO;

32, SEQ ID NO;

33, SEQ ID NO;

34, and SEQ ID NO;

35.

14. A method of controlling transcription of a nucleic acid sequence comprising inducing, in a cell comprising the nucleic acid construct of claim 2, the cis- regulator of translation.

15. The method of claim 14, wherein inducing is performed by contacting the cell with a nucleic acid that binds to the cis-regulator of translation of the nucleic acid construct.

16. A kit comprising:

(A) the nucleic acid construct of claim 2; and

(B) a regulatory input molecule, or a nucleic acid construct encoding a regulatory input molecule, that regulates the synthetic TCRT.