Bacteria one-hybrid vector and preparation

Abstract

The present invention relates to the field of genetic engineering technique, which is object to construct a bacterial single-hybrid vector, named pBXcmT, whose nucleotide sequence is shown as the sequence table SEQ ID NO: 1 and whose molecular weight is 3794563Da. The vector of the invention has highlight advantages that: it is fully compatible with the prior bacilli double-hybrid commercialization gene library, the report strain and other test reagents, in particular, it incorporates TA cloning, and can be easily and quickly used in the rapid cloning of short regulated nucleotide sequence. The bacterial single-hybrid vector has a wide range of applications in the identification and screening of DNA-protein interactions.

Description

technical field

[0001] The invention relates to a novel bacterial one-hybrid reporter carrier, which can be used for high-throughput bacterial one-hybrid screening so as to study specific DNA-protein interaction in Escherichia coli. Specifically, it relates to the preparation and application method of a bacterial single hybrid carrier. Background technique

[0002] Protein and nucleic acid are the two most important types of biomacromolecules that constitute life. The interaction between protein and nucleic acid is one of the central issues in molecular biology research. With the completion of the Human Genome Project, the function of genes has become a hot research topic. Various important physiological processes of cells, including signal transduction, cell response to internal and external environmental changes, etc., are all linked by the interaction between proteins and other substances. Studying the interactions between biomacromolecules such as protein-protein and D...

Examples

Embodiment 1

[0030] Embodiment 1 Preparation of carrier of the present invention

[0031] 1. Obtain the reporter gene His3-aadA fragment

[0032] The total DNA of the bacterial two-hybrid system reporter strain XL1-Blue MRF'Kan (purchased from Stratagene) was used as the template, and Rpb-f (5'-TAGAGGATCCTTGTCGAAGATCTTCGACAAC-3' and Rpb-r (5'-GAACCTCGAGTTATTTGCCAACTACCTTAGTGATCTCG-3') were used as templates. Primers carried out PCR amplification PCR amplification, obtained the His3-aadA reporter gene fragment of about 1.7kb, the obtained PCR product was cloned into pBS-T vector (purchased from TIANGEN company), positive clone corresponding sample sequencing (by INVITROGEN sequencing ) to verify the correctness of the target PCR product.

[0033] The PCR process is as follows:

[0034] Reaction system: Reaction procedure:

[0035] 2×GC Buffer I 37.5μl 96℃ 5min;

[0036] 2.5mM dNTP 8μl 94℃ 50sec;

[0037] Rpb-f 2μl 57℃ 50sec;

[0038] Rpb-r 2μl 72℃ 2min;

[0039] Genomic DNA 2μl 30 cy...

Embodiment 2

[0076] Embodiment 2: Carry out the method for high-throughput bacterial one-hybrid screening with pBXcmT carrier (biological application embodiment)

[0077] 1. Preparation of screening medium:

[0078] The reagents used in the screening medium of the present invention were all purchased from Stratagene Company.

[0079] (1) Accurately weigh 7.5g of agar powder, adjust the volume to 380mL with distilled water, adjust the pH to 6.8-7.0, sterilize at 121°C for 20 minutes, and keep it warm for later use;

[0080] (2) When the agar is cooled to 70°C, quickly add 50mL 10×M9 salts and mix well;

[0081] (3) When the above mixture is cooled to 50°C, quickly add 67.5mL M9 media additive, 0.5mL chloramphenicol (34mg / mL), 0.5mL tetracycline (12.5mg / mL), 10mL 3-AT (1M), 1mL Streptomycin (8mg / mL), mix quickly;

[0082] (4) Quickly pour the uniformly mixed culture medium onto a plate to form a screening plate, and place the plate at 4°C for standby (storage time shall not exceed one mon...

Embodiment 3

[0087] Example 3: Functional verification of the initial carrier pRpb

[0088]According to the documents already published by the applicant (Jiang PX, Feng Y, He ZG Functional differentiation and cooperative interaction between two eukaryote-like archaeal Orc1 / Cdc6 proteins on thereplication origin. Biochem Biophys Res Commun. 2007 Dec 28; 364 (4): 945 -51), the specific interaction between the replication initiation protein Cdc6 of the extreme thermophilic archaea Sulfolobus solfataricus P2 and the replication origin Ori1, and the C-terminus of Cdc62 is responsible for this interaction. However, in vitro experiments showed that the protein (Cdc 62Δc) after deletion of the C-terminus of Cdc62 still retained part of the function of interacting with Ori1. Therefore, the present invention selects S. solfataricus P2 replication initiation protein Cdc61, Cdc62, Cdc62Δc (C-terminal deletion of Cdc62) gene and S. solfataricus P2 replication origin Ori1 sequence respectively cloned in...

technical field

[0001] The invention relates to a novel bacterial one-hybrid reporter carrier, which can be used for high-throughput bacterial one-hybrid screening so as to study specific DNA-protein interaction in Escherichia coli. Specifically, it relates to the preparation and application method of a bacterial single hybrid carrier. Background technique

[0002] Protein and nucleic acid are the two most important types of biomacromolecules that constitute life. The interaction between protein and nucleic acid is one of the central issues in molecular biology research. With the completion of the Human Genome Project, the function of genes has become a hot research topic. Various important physiological processes of cells, including signal transduction, cell response to internal and external environmental changes, etc., are all linked by the interaction between proteins and other substances. Studying the interactions between biomacromolecules such as protein-protein and D...

Examples

Embodiment 1

[0030] Embodiment 1 Preparation of carrier of the present invention

[0031] 1. Obtain the reporter gene His3-aadA fragment

[0032] The total DNA of the bacterial two-hybrid system reporter strain XL1-Blue MRF'Kan (purchased from Stratagene) was used as the template, and Rpb-f (5'-TAGAGGATCCTTGTCGAAGATCTTCGACAAC-3' and Rpb-r (5'-GAACCTCGAGTTATTTGCCAACTACCTTAGTGATCTCG-3') were used as templates. Primers carried out PCR amplification PCR amplification, obtained the His3-aadA reporter gene fragment of about 1.7kb, the obtained PCR product was cloned into pBS-T vector (purchased from TIANGEN company), positive clone corresponding sample sequencing (by INVITROGEN sequencing ) to verify the correctness of the target PCR product.

[0033] The PCR process is as follows:

[0034] Reaction system: Reaction procedure:

[0035] 2×GC Buffer I 37.5μl 96℃ 5min;

[0036] 2.5mM dNTP 8μl 94℃ 50sec;

[0037] Rpb-f 2μl 57℃ 50sec;

[0038] Rpb-r 2μl 72℃ 2min;

[0039] Genomic DNA 2μl 30 cy...

Embodiment 2

[0076] Embodiment 2: Carry out the method for high-throughput bacterial one-hybrid screening with pBXcmT carrier (biological application embodiment)

[0077] 1. Preparation of screening medium:

[0078] The reagents used in the screening medium of the present invention were all purchased from Stratagene Company.

[0079] (1) Accurately weigh 7.5g of agar powder, adjust the volume to 380mL with distilled water, adjust the pH to 6.8-7.0, sterilize at 121°C for 20 minutes, and keep it warm for later use;

[0080] (2) When the agar is cooled to 70°C, quickly add 50mL 10×M9 salts and mix well;

[0081] (3) When the above mixture is cooled to 50°C, quickly add 67.5mL M9 media additive, 0.5mL chloramphenicol (34mg / mL), 0.5mL tetracycline (12.5mg / mL), 10mL 3-AT (1M), 1mL Streptomycin (8mg / mL), mix quickly;

[0082] (4) Quickly pour the uniformly mixed culture medium onto a plate to form a screening plate, and place the plate at 4°C for standby (storage time shall not exceed one mon...

Embodiment 3

[0087] Example 3: Functional verification of the initial carrier pRpb

[0088]According to the documents already published by the applicant (Jiang PX, Feng Y, He ZG Functional differentiation and cooperative interaction between two eukaryote-like archaeal Orc1 / Cdc6 proteins on thereplication origin. Biochem Biophys Res Commun. 2007 Dec 28; 364 (4): 945 -51), the specific interaction between the replication initiation protein Cdc6 of the extreme thermophilic archaea Sulfolobus solfataricus P2 and the replication origin Ori1, and the C-terminus of Cdc62 is responsible for this interaction. However, in vitro experiments showed that the protein (Cdc 62Δc) after deletion of the C-terminus of Cdc62 still retained part of the function of interacting with Ori1. Therefore, the present invention selects S. solfataricus P2 replication initiation protein Cdc61, Cdc62, Cdc62Δc (C-terminal deletion of Cdc62) gene and S. solfataricus P2 replication origin Ori1 sequence respectively cloned in...

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