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Multi-fragment DNA assembly kit, multi-fragment DNA assembly method and application thereof

A kit and multi-fragment technology, applied in the field of synthetic biology, can solve the problems of low assembly success rate, long time-consuming and high cost, and achieve the effect of high success rate, speeding up synthesis rate and reducing cost.

Pending Publication Date: 2020-04-07
SUZHOU HONGXUN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the Gateway technology needs to construct an entry vector, which is time-consuming and expensive; the Golden Gate technology relies on type II restriction endonuclease and ligase, and is not suitable for the synthesis of sequences with the above enzymes, and needs to analyze the fragment sequence in advance Then assemble; while the traditional Gibson assembly technology is less efficient when performing multi-fragment assembly, especially for fragments with larger fragments, the assembly success rate is very low

Method used

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  • Multi-fragment DNA assembly kit, multi-fragment DNA assembly method and application thereof
  • Multi-fragment DNA assembly kit, multi-fragment DNA assembly method and application thereof
  • Multi-fragment DNA assembly kit, multi-fragment DNA assembly method and application thereof

Examples

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Embodiment 1

[0070] In vitro assembly of embodiment 1 large fragment DNA

[0071] The construction process of large fragment recombinant plasmids is as follows: figure 1 As shown, in this example, the in vitro assembly of large fragments of DNA was first carried out.

[0072] (1) Splitting of large fragments of DNA

[0073] Use sequence analysis software to analyze large fragments of DNA with a length of 20 kb (such as image 3 ) for analysis, split into 10 initial fragments with a length of 2kb (such as image 3 ), and design homology arms between adjacent initial fragments (such as image 3 ) (homologous arms are the leftmost 60bp sequence and the rightmost 60bp sequence of the initial fragment), so that subsequent homologous recombination can be performed;

[0074] (2) Splitting of the initial segment

[0075] Use sequence analysis software to split the initial fragments into DNA fragments to be assembled F n , n=20, where, F n It is a long primer sequence separated by forward an...

Embodiment 2

[0089] Example 2 Preparation of Saccharomyces cerevisiae Electrotransfer Competent Cells

[0090] (1) Cultivate Saccharomyces cerevisiae overnight, reach the stable phase, and the OD600 reaches 3;

[0091] (2) Inoculate part of the yeast culture solution into 100mL YPD medium (10g / L yeast nitrogen base, 20g / L peptone, 20g / L D- -glucose), make the OD600 value reach 0.03, cultivate the yeast until the OD600 reaches 1.6, and collect the thalline by centrifugation;

[0092] (3) Wash the yeast pellet once with 50mL cold water, transfer it to a 20mL 0.1M LiAc / 10mM DTT Erlenmeyer flask, and treat it for 30min at 30°C and 225rpm on a shaking table;

[0093] (4) Wash the yeast pellet twice with 50 mL of cold water and once with 50 mL of 1M sorbitol;

[0094] (5) Suspend the yeast pellet in 100-200mL 1M sorbitol, then centrifuge and concentrate to 1mL, so that the concentration of yeast is equivalent to 1.6×10 9 Cells / mL, aliquoted for storage.

Embodiment 3

[0095] In vivo assembly of embodiment 3 large fragment DNA

[0096] Purify the in vitro assembly product obtained in Example 1 through a column, take 10 μL of the purified product and add it to Saccharomyces cerevisiae electroporation competent cells, mix well, set the electroporation program

[0097] The mixture of the purified large-fragment DNA and the assembled vector was electroporated into Saccharomyces cerevisiae competent cells by electroporation with a BioRad GenePulser cuvette (the distance between the electrodes is 0.2 cm), and the shock duration was 5 ms;

[0098] After electroporation, Saccharomyces cerevisiae cells were inoculated in 1 mL of sorbitol:YPD (1:1) medium with a concentration of 1 M, and cultured in suspension at 30°C for 1 h;

[0099] The collected cells were inoculated on the screening marker plate and cultured at 30°C for 3 days. The colony diagram is as follows: Figure 5 shown;

[0100]Pick a single colony with a sterilized toothpick and place ...

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Abstract

The invention provides a multi-fragment DNA assembly kit, a multi-fragment DNA assembly method and application thereof. The kit comprises a to-be-assembled DNA fragment Fn and a vector. The vector includes an intermediate connection vector and / or an assembly vector; the kit further comprises saccharomyces cerevisiae. According to the invention, a mode of combining in-vitro assembly and in-vivo assembly is adopted; an initial fragment is synthesized through PCR and the initial fragment is added into the intermediate connection vector, a recombinant enzyme system is adopted to assemble large-fragment DNA and the assembly vector in vitro, and then an in-vitro assembly product is converted into saccharomyces cerevisiae for in-vivo assembly. Advantages of in-vitro assembly and in-vivo assemblyare combined and the synthesis success rate of super-large plasmids is remarkably increased.

Description

technical field [0001] The invention belongs to the technical field of synthetic biology, and relates to a multi-segment DNA assembly kit, a multi-segment DNA assembly method and applications thereof, in particular to a multi-segment DNA assembly kit, a multi-segment DNA assembly method and its use in building large fragments Application of recombinant plasmids. Background technique [0002] In recent years, synthetic biology has developed rapidly and has been widely used in the fields of biomedicine, energy and new materials. Ultra-large plasmids can serve large-scale metabolic pathway research, chromosome research and genome research, and are helpful for gene function, metabolic pathway function research and understanding of life. However, there are many difficulties in the synthesis of super-large plasmids. Although scientists have proposed a variety of methods for the synthesis of super-large plasmids, there is generally a problem of low efficiency. This requires impro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/66C12R1/865
CPCC12N15/81C12N15/66
Inventor 朱佑民杨平孙健柳伟强刘敏祥严成丽邢妍婧赵一凡
Owner SUZHOU HONGXUN BIOTECH CO LTD
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