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Shuttle expression vector with broad hosts

A technology of host bacteria and sequence listing, applied in the direction of using vectors to introduce foreign genetic material, microorganism-based methods, microorganisms, etc., can solve the problems of limited application scope, slow growth, low cell yield, etc., to achieve a wide range of host capacity, high Conjugation transfer frequency, effect of stable replication

Active Publication Date: 2014-08-20
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the actual application process, the bacteria has the disadvantages of slow growth, low cell yield and poor adaptability to the metallurgical environment, which seriously limits its application range.

Method used

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  • Shuttle expression vector with broad hosts
  • Shuttle expression vector with broad hosts
  • Shuttle expression vector with broad hosts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Construction of Shuttle Expression Vector (Recombinant Plasmid pLAtcE)

[0037] 1. Construction of plasmid pLAtc-Km r

[0038]1. Synthesize the double-stranded DNA molecule (8144bp) shown in sequence 1 of the sequence listing. In sequence 1 of the sequence listing, the 81st to 755th nucleotides from the 5' end are the reverse complementary sequence of the gene encoding the replication protein RepC, and the 946th to 1803rd nucleotides are the reverse sequence of the encoding gene of the replication protein RepA Complementary sequence, the 2041st to 2530th nucleotide is the origin of replication oriV, the 2461st to 3105th nucleotide is the reverse complementary sequence of the coding gene of the junction protein MobE, and the 3065th to 3748th nucleotide is the junction protein MobD The reverse complementary sequence of the coding gene, the 3761st to 4159th nucleotide is the reverse complementary sequence of the coding gene of the binding protein MobC, the 423...

Embodiment 2

[0058] Example 2. Application of shuttle expression vector (recombinant plasmid pLAtcE)

[0059] 1. Construction of recombinant plasmid pLAtcE-eGFP

[0060] 1. Synthesize the double-stranded DNA molecule (eGFP gene) shown in sequence 8 of the sequence listing.

[0061] 2. Using the double-stranded DNA molecule synthesized in step 1 as a template, perform PCR amplification with a primer pair composed of F4 and R4 to obtain a PCR amplification product.

[0062] F4: 5'-CCC GAATTC ATGGTGAGCAAGGGCGCCGAGCTGTTC-3';

[0063] R4: 5'-CCC AAGCTT TCACTTGTACAGCTCATCCATGCCGTG-3'.

[0064] 3. Digest the PCR amplification product of step 2 with restriction endonucleases EcoRI and HindIII, and recover the digested product.

[0065] 4. Digest the recombinant plasmid pLAtcE with restriction endonucleases EcoRI and HindIII to recover the vector backbone.

[0066] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain the recombinant plasmid pLAtcE-eGFP. Acc...

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Abstract

The invention discloses a shuttle expression vector with broad hosts and provides a plasmid. The plasmid comprises the following components: a replication origin oriV, a gene for encoding replication protein Rep, a conjugation origin oriT, a gene for encoding conjugated protein Mob, an antibiotic selective marker gene and a foreign gene expression cassette, wherein the foreign gene expression cassette sequentially comprises a promoter, a multiple cloning endonuclease site in which a foreign gene is inserted, and a transcription termination sequence from upstream to downstream. The plasmid has a broad host ability, can be stably replicated in hosts and has high transconjugative frequency. The shuttle expression vector has great value for the genetic transformation of Acidithiobacillus caldus.

Description

technical field [0001] The invention relates to a shuttle expression vector with a wide range of hosts. Background technique [0002] Biometallurgy is one of the very active disciplines in the field of metallurgy in the past two decades, and has achieved industrial applications in the extraction of copper, gold, uranium and other metals. Biometallurgy is a technology that oxidizes, reduces or complexes metal minerals through the use of microorganisms, allowing metal ions to enter the solution for further separation, enrichment, and purification to extract metals. Compared with conventional physical and chemical dressing and smelting methods, biometallurgy has outstanding advantages such as simple process, low cost, low energy consumption, and little environmental pollution. , Heap leaching and in-situ leaching of refractory ores. In today's situation where there is a large demand for resources, the shortage of easy-to-develop resources, and the continuous improvement of en...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74C12R1/01
Inventor 刘双江张明江姜成英
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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