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Method of assembling multiple gene fragments to py-2u vector via yeast and application

A technology of py-2u and gene fragments, applied in the field of bioengineering, can solve the problems of high mutation rate, time-consuming and laborious assembly of small fragments, difficulty in amplifying fragments above 10kb by polymerase chain reaction, etc., and achieve simple, convenient and high-speed cloning process Stirring and centrifugation, time-saving effect

Active Publication Date: 2019-08-16
通用生物(安徽)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The traditional large-fragment cloning technology to obtain large fragments of interest has various defects. For example, random library construction and cloning needs to rely on high-throughput screening; polymerase chain reaction (PCR) is difficult to amplify fragments above 10kb, and assembling small fragments is time-consuming and laborious. High mutation rate; difficult to find suitable restriction endonuclease sites at both ends of the fragment based on restriction ligation

Method used

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  • Method of assembling multiple gene fragments to py-2u vector via yeast and application
  • Method of assembling multiple gene fragments to py-2u vector via yeast and application
  • Method of assembling multiple gene fragments to py-2u vector via yeast and application

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Experimental program
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Embodiment 1

[0042] refer to Figure 1-6 As shown, the method of using yeast to assemble multiple gene fragments into the py-2u vector specifically includes the following steps:

[0043]S1. Add a homologous sequence homologous to the py-2u vector at the two segments of the target gene to be cloned, with a length of 40-60bp;

[0044] S2. Divide the gene obtained in step S1 into a plurality of small fragments, which can usually be divided equally according to the length, and a separate enzyme cutting site is added to the two segments (this enzyme cutting site should not exist in the target gene, otherwise the enzyme will be used later) When a small fragment plasmid is digested with a cleavage site, the target gene containing the cleavage site will be cut). The restriction sites at both ends of each small fragment are the same restriction site, or different individual restriction sites.

[0045] S3. Each small fragment can be obtained by gene synthesis or by PCR amplification, and the blunt...

Embodiment 2

[0054] refer to figure 1 As shown, 3 vector plasmids were randomly selected, extracted after arabinose induction, and electrophoresed on the plasmids, see lanes 1, 2, and 3, and electrophoresed in lanes 4, 5, and 6 after extraction of plasmids in the uninduced control group, visible lanes The concentration of uninduced plasmid was lower, and the content of supercoiled plasmid was lower than that of induced plasmid. The values ​​measured with a onedrop spectrophotometer are as follows:

[0055]

[0056] refer to figure 2 Shown is the final restriction map of the plasmid, digested with AscI and PmlI, the correct size is 9k / 5.2k. The time recording results of related operations are shown in the table below:

[0057]

[0058]

[0059] In this example, yeast can be used to assemble 8 gene fragments at a time, which greatly exceeds the upper limit of the fragments that can be assembled by traditional assembly methods (2-3 gene fragments, even if it is Gibson, the succes...

Embodiment 3

[0063] refer to Figure 3-6 , This embodiment provides a constant temperature incubation device, which can accommodate, centrifugally stir, and heat various test tubes and glass slides of different specifications and sizes. Specifically, the constant temperature incubation device includes a thermal insulation shell 100, an airtight door 200, and accommodates a centrifugal mechanism. The interior of the thermal insulation shell 100 has a cylindrical cavity, and the airtight door 200 is used to accommodate the opening and closing of the centrifugal mechanism. The top of the thermal insulation shell 100 is hinged, and the airtight door 200 is connected with the bottom of the thermal insulation shell 100 with a cylinder telescopic rod 300 .

[0064] The centrifuge accommodating mechanism is arranged in the cylindrical cavity inside the thermal insulation shell 100, including: a shock-absorbing disc 400, a shock-absorbing installation mechanism, and a multi-test tube accommodating ...

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Abstract

The invention discloses a method of assembling multiple gene fragments to a py-2u vector via a yeast and application and relates to the technical field of bioengineering. Homologous sequences homologous to the py-2u vector are added to the two ends of a target gene to be cloned; the obtained gene is divided into multiple small fragments; independent digestion sites are added to two ends of each fragment; the digestion sites at the two ends of each fragment are identical, or different independent ones; each small fragment is acquired through genetic synthesis or PCR (polymerase chain reaction)amplification; the plain end of each amplified product is connected to a cloning vector or is TA-cloned to one small fragment vector; each fragment is subjected to enzyme digestion via one independentdigestion site; the recycled product is applied to next yeast transformation. The constructed Py-2uL may be induced via arabinose to gain a larger copy number in Escherichia coli, so that genetic operations, such as subsequent plasmid extraction, are facilitated; genetic synthesis and assembly of a growth gene can be achieved stably and quickly; the method is simpler than conventional cloning processes and is economical and efficient.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method and application of using yeast to assemble multiple gene fragments into a py-2u vector. Background technique [0002] Synthetic biology has great potential in the fields of biomedicine, biofuel, fine chemicals, and agriculture. With the rapid development of synthetic biology, it has gradually entered the era of whole genome synthesis, and the technology of synthetic genomics is of great significance. DNA genome cloning and assembly technology is an important molecular biology tool, which plays a key role in the rapid and efficient assembly of large fragments of DNA elements. DNA synthesis is the core technology supporting the development of synthetic biology. It does not depend on DNA templates and can be directly synthesized based on known DNA sequences. Play an important role in the artificial synthesis of genomes. [0003] The patent application number 201310...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/66
CPCC12N15/66C12N15/81
Inventor 雍金贵喻明军苏万凯施荣辉王维昆
Owner 通用生物(安徽)股份有限公司
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