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Plasmid construction kit, plasmid construction method and application thereof

A kit and plasmid technology, applied in the field of synthetic biology, can solve the problems of weak assembly ability of large fragments of DNA, difficult in vivo assembly of super-large plasmids, low assembly success rate, etc. The effect of the synthesis success rate

Pending Publication Date: 2020-04-10
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
[0004] The yeast in vivo assembly method is limited by the growth rate of yeast, which takes a long time; Escherichia coli grows fast, but its ability to assemble large fragments of DNA is weak, and it is difficult to complete the in vivo assembly of super large plasmids

Method used

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  • Plasmid construction kit, plasmid construction method and application thereof
  • Plasmid construction kit, plasmid construction method and application thereof
  • Plasmid construction kit, plasmid construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0072] In vitro assembly of embodiment 1 plasmid

[0073] The plasmid construction process is as follows figure 1 As shown, in this example, the in vitro assembly of plasmids was first carried out.

[0074] (1) Splitting of large fragments of DNA

[0075] Use sequence analysis software to analyze the 10kb large fragment DNA, split it into five initial fragments with a length of 2kb, and design homology arms between adjacent initial fragments (homology arms are the leftmost 60bp sequence of the initial fragment and the rightmost 60bp sequence) sequence) for subsequent homologous recombination;

[0076] (2) Splitting of the initial segment

[0077] Use sequence analysis software to split the initial fragments into DNA fragments to be assembled F n , n=30, F n It is a long primer sequence separated by forward and reverse, and the adjacent fragment contains a 20bp overlapping reverse complementary sequence;

[0078] (3) PCR synthesis of initial fragments

[0079] Use PCR to...

Embodiment 2

[0091] Example 2 Preparation of Escherichia coli Electrotransfer Competent Cells

[0092] (1) Pick fresh EPI300 colonies and inoculate them into 4 mL of LB medium, and culture overnight at 37°C under a well-ventilated condition;

[0093] (2) Dilute 1 mL of overnight cultured cells into 300 mL of LB medium, and culture at 37°C for 2-3 hours in a well-ventilated condition until the OD600 is 0.5;

[0094] (3) Collect cells by centrifugation at 3,500rpm for 10min;

[0095] (4) Wash the cells twice with ice-cold sterile washing buffer, and centrifuge at 3,500 rpm for 10 min;

[0096] (5) The cell suspension was centrifuged at 3,500 rpm for 10 minutes, and the supernatant was poured out immediately after the centrifugation;

[0097] (6) Wash the cells twice with a solution containing 10% ultrapure glycerol and 90% ultrapure double distilled water, and centrifuge at 4,000 rpm for 10 min;

[0098] (7) Resuspend the cells in washing buffer (approximately 2 mL), aliquot and freeze at...

Embodiment 3

[0099] Example 3 Preparation of Escherichia coli containing λ phage Red recombination system

[0100](1) Transform the plasmid pKD46 containing the λ phage Red recombination system into Escherichia coli electroporation competent;

[0101] (2) Cultivate at 30°C, select antibiotics to successfully transform Escherichia coli with pKD46;

[0102] (3) Escherichia coli containing pKD46 was made into competent cells.

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Abstract

The invention provides a plasmid construction kit, a plasmid construction method and application thereof. The kit comprises a to-be-assembled DNA fragment Fn and a carrier, wherein the carrier comprises an intermediate carrier and / or an assembly carrier; the kit also comprises escherichia coli, and the escherichia coli contains plasmids of a lambda phage Red recombination system. An in-vitro assembly and in-vivo assembly combined mode is adopted by the invention, firstly a recombinase system is employed to assemble large-fragment DNA and the assembly carrier in vitro, and then the in-vitro assembly product is transferred into escherichia coli containing the lambda phage Red recombinant system plasmids for in-vivo assembly, the constructed kit combines the advantages of in-vitro assembly and in-vivo assembly, also the lambda phage Red recombination system is employed to enhance the in-vivo assembly capacity of escherichia coli, the plasmid synthesis rate is accelerated, and the plasmidsynthesis success rate is remarkably increased.

Description

technical field [0001] The invention belongs to the technical field of synthetic biology, and relates to a plasmid construction kit, a plasmid construction method and applications thereof. 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 be used to synthesize structural units of the genome, serve technologies such as gene editing, protein expression, and antibody expression, and are helpful for research on metabolic pathways, gene functions, protein functions, and immunotherapy. However, there are many difficulties in the synthesis of super-large plasmids, especially those above 40kb. 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 improvements and breakthroughs in the synthesis and assembly of super-large plasmids. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/64
CPCC12N15/70C12N15/64
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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