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A method for the rapid assembly of multi-fragmented DNA in yeast

An assembly method and multi-fragment technology, applied in the field of synthetic biology, can solve the problems of reduced fidelity, increased operational complexity, increased workload, and prolonged overall time, achieving low cost, wide application, and high success rate.

Active Publication Date: 2019-01-11
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the existing yeast recombination technology needs to select yeast single colony culture and screen one by one, and the linearized yeast vector must be obtained by PCR (Gibson, DG.et.al.Science (2008) 5867:1215-1220), and the overall time will be corresponding extension, the fidelity will decrease, and the complexity and workload of the operation will increase

Method used

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  • A method for the rapid assembly of multi-fragmented DNA in yeast
  • A method for the rapid assembly of multi-fragmented DNA in yeast
  • A method for the rapid assembly of multi-fragmented DNA in yeast

Examples

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Embodiment 1

[0044] Embodiment 1: a kind of yeast quick assembly method of multi-fragment DNA, comprises the following steps:

[0045] (1) Co-transform yeast with multiple DNA molecules containing homology arms and linearized yeast shuttle vector:

[0046]① Use K410F shown in SEQ ID NO.1 as the upstream primer, K410R shown in SEQ ID NO.2 as the downstream primer, and pEasy‐K410 shown in SEQ ID NO.3 as the template, using Phusion high-fidelity DNA polymerase Perform PCR to obtain DNA molecular fragment K410; K411F shown in SEQ ID NO.4 is used as an upstream primer, K411R shown in SEQ ID NO.5 is used as a downstream primer, and pEasy‐K411 shown in SEQ ID NO.6 is used as a template, Use Phusion high-fidelity DNA polymerase to perform PCR to obtain DNA molecular fragment K411; use K412F shown in SEQ ID NO.7 as the upstream primer, and K412R shown in SEQ ID NO.8 as the downstream primer, shown in SEQ ID NO.9 The pEasy-K412 of the Phusion high-fidelity DNA polymerase was used as a template to o...

Embodiment 2

[0067] Embodiment 2: a yeast rapid assembly method of multi-fragment DNA, comprising the following steps:

[0068] (1) Co-transform yeast with multiple DNA molecules containing homology arms and linearized yeast shuttle vector:

[0069] ① Use tetR-F shown in SEQ ID NO.19 as the upstream primer, tet-R shown in SEQ ID NO.20 as the downstream primer, and ptetR shown in SEQ ID NO.21 as the template, using Phusion high-fidelity DNA Polymerase performs PCR to obtain the DNA molecular fragment TetR; VioAF shown in SEQ ID NO.22 is used as an upstream primer, VioAR shown in SEQ ID NO.23 is used as a downstream primer, and pVioA shown in SEQ ID NO.24 is used as a template, Use Phusion high-fidelity DNA polymerase to perform PCR to obtain the DNA molecular fragment VioA; VioBF shown in SEQ ID NO.25 is used as the upstream primer, and VioBR shown in SEQ ID NO.26 is used as the downstream primer, shown in SEQ ID NO.27 pVioB as a template, and use Phusion high-fidelity DNA polymerase to pe...

Embodiment 3

[0082] Embodiment 3: a kind of yeast rapid assembly method of multi-fragment DNA, comprises the following steps:

[0083] (1) Co-transform yeast with multiple DNA molecules containing homology arms and linearized yeast shuttle vector:

[0084] ①Using TEF1p-F shown in SEQ ID NO.38 as the upstream primer, TEF1p-R shown in SEQ ID NO.39 as the downstream primer, using the yeast genome as a template, and using Phusion high-fidelity DNA polymerase for PCR to obtain SEQ ID NO. The DNA molecule fragment TEF1p shown in ID NO.56; with PDX1t-F shown in SEQ ID NO.40 as the upstream primer, TDH3p-R shown in SEQ ID NO.41 as the downstream primer, using the yeast genome as a template, using Perform PCR with Phusion high-fidelity DNA polymerase to obtain the DNA fragment PDX1t-TDH3p shown in SEQ ID NO.57; use MPE1t-F shown in SEQ ID NO.42 as the upstream primer, and FBA1p shown in SEQ ID NO.43 ‐R is the downstream primer, using the yeast genome as a template, using Phusion high-fidelity DNA ...

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Abstract

The invention discloses a rapid assembling method of multi-fragment DNA yeast. The rapid assembling method comprises the following steps: (1) performing co-transformation on a plurality of DNA molecules with homologous arms, and linearized yeast shuttle vectors, so as to obtain yeast; (2) eluting all the transformed yeast colonies on a whole screening culture plate, centrifuging, and discarding the supernate to obtain transformed yeast cells; (3) extracting plasmid DNA of the yeast cells obtained in step (2), transforming the competent cells of colon bacillus; and (4) screening colon bacillus, and cloning to obtain large-fragment DNA. The method for assembling large-fragment DNA molecules is fast in speed, simple, convenient and feasible, high in success rate, low in cost, high in efficiency, easy to operate, beneficial in enlarging the industrialization scale and wide in application, and a plurality of small-fragment DNA molecules can be assembled into one large-fragment DNA molecule.

Description

technical field [0001] The invention relates to the field of synthetic biology, genome synthesis technology, and metabolic engineering field, in particular to a yeast rapid assembly method for multi-segment DNA, a large-segment DNA synthesized by the method and its application. Background technique [0002] The technology of synthesizing large fragments of DNA molecules is of great significance. On the one hand, it can be used to synthesize structural units of the genome, and on the other hand, it can be used to assemble metabolic pathways that are of great production and application value to humans. [0003] Today's biological development has gradually entered the era of whole genome synthesis. Synthetic genomics is of great significance for people to understand life and transform life to better serve human society. Genome synthesis is an important aspect of synthetic chassis cells. In terms of design: artificially design the growth function genes and expression function ge...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12N15/11C12N15/81C12P23/00C12P17/16
Inventor 元英进贾斌林秋卉张文倩张金来李炳志其他发明人请求不公开姓名
Owner TIANJIN UNIV
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