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PCCI-2U plasmid as well as construction method and application thereof

A PCCI-2U, plasmid technology, applied in the fields of biotechnology and genetic engineering, can solve the problems of plasmid extraction difficulties, sequence deletion, capacity limitation, etc., and achieve the effect of low copy number, large capacity and high stability

Pending Publication Date: 2020-11-06
江苏赛索飞生物科技有限公司
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Problems solved by technology

[0008] The problems associated with the cloning of large fragments of DNA include: conventional vectors have limited capacity for cloning large fragments of DNA, and sequence deletions and cloning failures often occur when cloning genes of more than 20k
Therefore, compared with conventional cloning vectors, the plasmid extraction of BAC vectors is relatively more difficult, because the plasmid copy number is several tenths of conventional

Method used

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  • PCCI-2U plasmid as well as construction method and application thereof
  • PCCI-2U plasmid as well as construction method and application thereof
  • PCCI-2U plasmid as well as construction method and application thereof

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Embodiment

[0037] Construction of plasmid PCCI-2U:

[0038] The structure of plasmid PCCI-2U is as follows figure 1 As shown, the construction process pattern diagram is as follows figure 2 As shown, the specific method is as follows:

[0039] (1) Design and synthesize the gene sequence of Trp-2μ gene. As shown in sequence 1 and sequence 3 in the sequence table, the length of sequence 1 is 957bp, which is a trp gene, encoding tryptophan synthase, and the length of sequence 3 is 1343bp, which is a 2μ replicon . The sequence and upstream and downstream sequences have a homologous sequence of 30bp.

[0040] (2) Designing and synthesizing the Cm-oriⅤ-ori2 gene, the gene sequence is shown in sequence 2 in the sequence table, the full length of the sequence is 2530bp, wherein 1-763 is a chloramphenicol resistance gene, encoding chloramphenicol acetyltransferase, 1620-2235bp is the oriⅤ replicon gene, 2310-2530bp is the ori2 replicon gene;

[0041] (3) Design and synthesize the repE-SopA-...

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Abstract

The invention relates to a PCCI-2U plasmid as well as a construction method and application thereof. The plasmid is named as PCCI-2U and comprises a repE gene, a sopA gene, a sopB gene, a sopC gene, an ori2 replicon, an oriV replicon, a prokaryotic transcription terminator T3te, a tonB terminator, a trp gene and a yeast 2mu replicon gene. The invention provides the PCCI-2U plasmid as well as the construction method and the application thereof. The plasmid has the characteristics of low copy number, large capacity, high stability and the like in escherichia coli, can be stably copied by depending on a high-copy 2mu replicon in yeast, has the TRP gene for encoding tryptophan, and can be screened by utilizing an SC-rtp semi-synthetic culture medium. The PCCI-2U disclosed by the invention canbe assembled in yeast with long genes, and is applied to the gene engineering fields of cloning, screening, sequencing and genome construction of long-fragment genes and complex-structure genes.

Description

technical field [0001] The invention belongs to the field of biotechnology and genetic engineering, and specifically relates to a construction method and application of a large-capacity, high-stability yeast-large intestine shuttle vector PCCI-2U plasmid capable of switching between single copy and medium copy. Background technique [0002] Since the establishment of modern biology, from the Human Genome Project that decodes a person's genome project, to HapMap that analyzes population differences, to GWAS that uses genomes to suppress diseases, and now precision medicine, among the huge treasures of the genome, human Exploration never stops. Scientists now have the ability to construct or reprogram the genomes of E. coli or yeast from scratch. [0003] Genome assembly technology is based on large-scale genome data analysis to discover new or hidden bioactive substance synthesis gene clusters. Using gene assembly technology, the expression of silent biosynthetic gene clust...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/69C12N15/65
CPCC12N15/81C12N15/69C12N15/65C12N2800/30
Inventor 开雷杜攀苏万凯
Owner 江苏赛索飞生物科技有限公司
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