Granted Patent B2

US 12,570,989 · App. 16/914,266 · Granted Mar 10, 2026

Inventors: Gaoxu Xue (Suzhou, CN); Tianming Qi (Suzhou, CN); Aihua Feng (Suzhou, CN); Zhengli Xie (Suzhou, CN); Yankai Jia (Suzhou, CN); Xin Wu (Suzhou, CN); Zhongping Sun (Suzhou, CN); Guojuan Liao (Suzhou, CN)

Assignee: AZENTA US, INC.

C12N15/66C12N9/2471C12N15/70C12N15/72C12Y302/01023C12N2800/22C12N2830/55

Abstract

Claims (29)

1 . A β-galactosidase promoter, consisting of a −35 region, a −10 region, and a mutated nucleic acid sequence between the −35 region and the −10 region, wherein:

the mutated nucleic acid sequence comprises at least one recognition site for an endonuclease, each recognition site for an endonuclease selected from the group consisting of EcoRV, AleI, BamHI, XhoI, and PmlI;

the −35 region has a nucleic acid sequence of 5′-TTTACA-3′;

a 5′ end of the mutated nucleic acid sequence is linked to a 3′ end of the −35 region;

a 3′ end of the mutated nucleic acid sequence is linked to a 5′ end of the −10 region; and

the mutated nucleic acid sequence is 16 to 18 base pairs in length.

2 . The promoter of claim 1 , wherein the mutated nucleic acid sequence between the −35 region and the −10 region of the promoter is SEQ ID NO: 4.

3 . A vector, comprising the promoter of claim 1 and a reporter gene regulated by the promoter.

4 . The vector of claim 3 , wherein insertion of a foreign DNA at the recognition site for the endonuclease significantly reduces activity of the promoter.

5 . The vector of claim 3 , wherein the vector is a cloning vector or an expression vector.

6 . An isolated host cell, comprising the vector of claim 3 .

7 . The host cell of claim 6 , wherein the host cell is Escherichia coli.

8 . The host cell of claim 7 , wherein a C-terminal ω-fragment of a β-galactosidase of the Escherichia coli is encoded.

9 . A method of preparing the vector of claim 3 , comprising the following steps:

(1) performing PCR with a set of primers for mutating a nucleic acid sequence between the −35 region and the −10 region of a β-galactosidase promoter in a promoter region of a β-galactosidase to comprise at least one recognition site for an endonuclease, and using an original vector comprising an original β-galactosidase promoter and a reporter gene regulated by the original β-galactosidase promoter as a template for PCR amplification, to obtain a PCR amplification product with the promoter of claim 1 ; and

(2) cyclizing the PCR amplification product obtained using the primers in step (1) by recombination to obtain a vector.

10 . The method of claim 9 , wherein nucleic acid sequences of the set of primers in step (1) are SEQ ID NO: 17 and SEQ ID NO: 18.

11 . A kit, comprising either a vector comprising the promoter of claim 1 or an isolated host cell comprising the vector.

12 . The method of claim 9 , wherein before step (1), the method further comprises performing codon optimization on the reporter gene.

13 . The method of claim 9 , wherein the reporter gene is a lacZ gene whose nucleic acid sequence is SEQ ID NO: 39.

14 . The vector of claim 3 , wherein the reporter gene is a lacZ gene whose nucleic acid sequence is SEQ ID NO: 39.

15 . The method of claim 9 , wherein the reporter gene is a lacZ gene encoding an N-terminal α-fragment of the β-galactosidase.

16 . The promoter of claim 1 , wherein the −10 region has a nucleic acid sequence of 5′-TATGTT-3′.

17 . The vector of claim 4 , wherein the foreign DNA is at least 24 base pairs in length.

18 . A method of using the vector of claim 3 , comprising the following steps:

(1) linearizing the vector by digesting the at least one recognition site for the endonuclease in the mutated nucleic acid sequence between the −35 region and the −10 region of the promoter to obtain a linearized vector; and

(2) ligating a foreign DNA into the linearized vector of step (1), thereby obtaining a vector comprising a foreign DNA inserted into the mutated nucleic acid sequence between the −35 region and the −10 region of the promoter.

19 . The method of claim 18 , wherein the linearizing in step (1) is performed through endonuclease digestion and PCR amplification.

20 . The method of claim 18 , wherein digesting is performed with an appropriate endonuclease selected from the group consisting of EcoRV, AleI, BamHI, XhoI, and PmlI.

Assignments (8)

Priority Claims (1)

Continuity (2)

References Cited (106)