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Shuttle vector beneficial to construction of long fragment DNA and preparing method and application thereof

A technology of shuttle vectors and long fragments, applied in recombinant DNA technology, biochemical equipment and methods, vectors, etc., can solve problems such as inability to construct, plasmids that cannot meet sequencing requirements, etc., to reduce production costs, reduce workload, improve copy number effect

Pending Publication Date: 2017-03-15
SUZHOU HONGXUN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It cannot be constructed by conventional recombination-based methods, such as Gibson recombination, In-Phusion recombination, etc.
In the yeast system, the assembled fragments are relatively large, and a low-copy plasmid is required to ensure its stability. However, due to the thick cell wall of yeast, the proposed plasmid cannot meet the sequencing requirements.

Method used

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  • Shuttle vector beneficial to construction of long fragment DNA and preparing method and application thereof
  • Shuttle vector beneficial to construction of long fragment DNA and preparing method and application thereof
  • Shuttle vector beneficial to construction of long fragment DNA and preparing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Preparation of shuttle carrier:

[0055] 1. The four fragment sequences shown in SEQ ID NO.13 to 16 were respectively obtained by gene synthesis method and two rounds of PCR amplification, specifically:

[0056] Step (1), add 5ul of each 20pM / ul primer, mix with 100ul ddH 2 O dilution, the first round of PCR reaction system is:

[0057] High-fidelity PCR enzyme 1ul dNTPs 1ul 5X high fidelity PCRBuffer 10ul Primer 1ul tail primer 1ul Primer Mix 10ul wxya 2 o

TO 50ul

[0058] The first round of PCR reaction procedure is:

[0059]

[0060] Step (2), with the first round of PCR reaction solution as template, carry out the second round of PCR amplification, the reaction system of the second round of PCR amplification is:

[0061] S15 enzyme 1ul dNTPs 1ul 5X high fidelity PCRBuffer 10ul Primer 1ul tail primer 1ul Primer Mix 1ul wxya 2 o

TO50ul

[0...

Embodiment 2

[0074] (1), Carry out enzyme digestion and fragment amplification to the carrier prepared in Example 1: ASCⅠ is selected for the enzyme digestion site, the overlaps between the fragment and the carrier, and the fragment and the fragment are at 80bps, and the sequence of each fragment is as SEQ ID NO .2 to SEQ ID NO.11. The vector was digested with ASCⅠ, and the fragment was amplified by PCR.

[0075] (2) Precipitation of fragments and linearized vectors

[0076] Take 3ul of each fragment of the obtained linearized plasmid and fragment, precipitate with isopropanol in equal volume and add 12% NaAc, precipitate at minus 25°C for 1.5h, centrifuge at 13,000 rpm for 10min at room temperature, discard the supernatant, and wash with 70 Wash twice with 500ul of % ethanol, discard the supernatant, blow in a biological safety cabinet for 10min, and dissolve with 20ul sterile water.

[0077] (3), preparation of yeast electroporation competent cells

[0078] a. The competent Saccharomy...

Embodiment 3

[0098] (1), carry out enzyme digestion and amplification of fragments to the carrier prepared in Example 1: EcoR Ⅰ is selected as the enzyme digestion site, fragments and vectors, overlaps between fragments and fragments are at 80bps, and the sequence of each fragment is as SEQ ID NO.43 to SEQ ID NO.52. The vector was digested with EcoR Ⅰ, and the fragment was amplified by PCR.

[0099] (2) Precipitation of fragments and linearized vectors

[0100] Take 3ul of each fragment of the obtained linearized plasmid and fragment, precipitate with isopropanol in an equal volume and add one-tenth volume of NaAc, precipitate at 20°C for 1h, 13,000 rpm, room temperature, 10min, discard the supernatant, and use Wash twice with 500ul of 70% ethanol, discard the supernatant, blow in a biological safety cabinet for 10min, and dissolve with 20ul sterile water.

[0101] (3), preparation of yeast electroporation competent cells

[0102] a. The competent Saccharomyces cerevisiae solution to be...

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Abstract

The invention relates to a shuttle vector beneficial to construction of long fragment DNA and a preparing method and application thereof. The shuttle vector is capable of shuttling back and forth in saccharomyces cerevisiae and escherichia coli, and has a saccharomyces cerevisiae replication origin oriV and an escherichia coli replication origin ori2, both the oriV and the ori2 are low-copy replication origins, and the shuttle vector can conduct stable replication in both the saccharomyces cerevisiae and the escherichia coli, a screening label in the saccharomyces cerevisiae is HIS3, and the screening label in the escherichia coli is chlorampenicol resistance. According to the shuttle vector beneficial to the construction of long fragment DNA and the preparing method and application thereof, the constructed saccharomyces cerevisiae-escherichia coli shuttle vector (named pSynoYAC0) is capable of being successfully assembled in a yeast system, however, plasmid extracted from the saccharomyces cerevisiae has poor quality, while the plasmid extracted from the escherichia coli has high quality, thus further experiment demands such as sequencing and enzyme digestion can be satisfied. The shuttle vector is also capable of conducting replication in the escherichia coli, and the replication belongs to a single copy; meanwhile, the shuttle vector is capable of existing stably in the replication process, thus the vectors meeting the demands can be obtained.

Description

technical field [0001] The invention relates to a shuttle carrier which is beneficial for constructing long-segment DNA, its preparation method and application. Background technique [0002] With the development of basic synthetic biology, research in the field of life sciences requires comprehensive analysis of complex pathways in vitro, construction of new biological components, and redesign of natural biological systems. At present, in vitro assembly technologies mainly include Gateway, In-fusion, Cold-fusion, T4 DNA polymerase-based Ligase Independent Cloning (LIC), Golden Gate, Gibson Assembly, etc. Currently, Gibson Assembly is the most widely used technology, which has a wide range of applications , simple, effective, robust and fast, and can assemble 4 target fragments into the target carrier at one time, which has greater advantages compared with other in vitro assembly techniques. However, the technique is capable of building up to around 10kb. With the developme...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/64
CPCC12N15/70C12N15/64C12N2800/101C12N2820/55C12N2820/704
Inventor 郭天玮钟云鹏李彦敏杨平
Owner SUZHOU HONGXUN BIOTECH CO LTD
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