DNA construct and DNA extracorporeal splicing method

A technology of constructs and vectors, applied in the field of genetically modified constructs and DNA in vitro splicing, can solve the problems of limiting the versatility of bio-brick assembly technology, huge workload, and uncertain functions.

Inactive Publication Date: 2016-02-17
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

De novo synthesis is not only expensive and slow, but also the function of the DNA sequence may be affected after part of the DNA sequence is changed; and the synonymous mutation by PCR is not only a huge workload (especially for some larger biological bricks) blocks), and will also have uncertain effects on the function of the DNA
At the same time, this requirement limits the versatility of bio-brick assembly technology, making it impossible for some laboratories to perform hierarchical assembly of biological functional elements without removing the internal enzyme cleavage site.

Method used

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  • DNA construct and DNA extracorporeal splicing method
  • DNA construct and DNA extracorporeal splicing method
  • DNA construct and DNA extracorporeal splicing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] Example 1 Heterologous Expression of Lycopene Biosynthesis Gene Cluster

[0124] 1. Using pUC18 as a template, the bla gene and the replicon were amplified by PCR. There were PacI and NotI restriction sites at both ends of the bla gene, and NotI and SwaI-AscI sites at both ends of the replicon. Digest the two fragments with NotI (37°C / 1h), mix and connect the two fragments at an equimolar ratio, add T4PNK (T4PolynucleotideKinase) to the system during connection, connect at 16°C overnight, transform E. coli DH5α, add ampicillin antibiotics in LB Positive clones were screened and verified by sequencing. The correct plasmid was named pJK5.

[0125] 2. Using BBa_274100 as a template to amplify the crtEBI (crtE, crtB, crtI) gene; using pKD46 as a template to amplify the pBAD promoter. Among the above parts, the 5' end of crtE has SwaI and I-SceI sites, and the 3' end has AscI and PI-PspI sites; the 5' end of the other parts only has I-SceI sites, 3 ' ends with only PI-Psp...

Embodiment 2

[0131] Example 2 Heterologous Expression of Actinorhodine Biosynthetic Gene Cluster

[0132] 1. Use pET28a as a template to amplify the kanamycin resistance gene, then use PacI and NotI to double-enzyme digest and remove phosphorus, and recover; at the same time, use these two enzymes to excise the ampicillin resistance gene in pIB1A1_0-C000001, and then ligate Kana resistance gene was inserted, and the resulting plasmid was named pIB1K1_0-C000001. Using the same method, the abramycin resistance gene was amplified using the pSET152 plasmid as a template, and then the ampicillin resistance gene in pIB1A1_0-C000001 was replaced with the abramycin resistance gene to obtain the plasmid pIB1Am1_0-C000001.

[0133] 2. Digest pIB1A1_0-C000001 with SwaI and NotI and dephosphorize it as a carrier, then use these two enzymes to cut the replicon of pCC2FOS (see Example 1 for the preparation of the replicon), and obtain plasmid pIB2A1_0-C000001 after ligation. Digest pIB1K1_0-C000001 wit...

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Abstract

The invention provides a DNA construct containing a nucleic acid sequence shown as a structural formula as Ha-C-Hb, in the formula, Ha is a first homing endonuclease enzyme site; C is the nucleic acid sequence to be spliced; Hb is a second homing endonuclease enzyme site; wherein, and the first homing endonuclease enzyme site and the second homing endonuclease enzyme site are different kinds of endonucleases. The invention provides an application of the DNA construct and a DNA extracorporeal splicing method by using the DNA construct. The construct and the method can realize engineering, standardization, modularization reconstruction or assembly for DNA.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a construct for genetic modification and a method for splicing DNA in vitro. Background technique [0002] The breakthrough of DNA synthesis and assembly technology has greatly promoted the progress in the field of synthetic biology, and with the in-depth development of synthetic biology, more challenges and requirements have been put forward for DNA synthesis and assembly technology. The emergence of the standardized assembly technology of biological bricks has been widely used and welcomed by the synthetic biology community, which greatly simplifies the thinking design and workload in the process of constructing genetic circuits. [0003] The core concept of synthetic biology "engineering, standardization, modularization" in BioBricks (BioBricks TM ) This splicing standard has been well reflected and disseminated. In particular, the IGEM international compe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63C12N15/10C12N1/21C12N1/15C12N1/19C12N5/10
Inventor 王金赵国屏刘家坤陈威华
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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