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Design and construction methods and application of standardized bio-elements

A construction method and biological technology, applied in the fields of metabolic engineering, synthetic biology, and genetic engineering, can solve the problems of huge cost, difficulty in constructing and optimizing exogenous metabolic pathways, and inability to directly apply yeast and other eukaryotic microorganisms.

Active Publication Date: 2016-11-23
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the nitrogen fixation pathway in Klebsiella oxytoca consists of 20 genes, and there are many unknown regulatory factors, so it is difficult to construct and optimize the entire exogenous metabolic pathway
On the other hand, although the development of molecular biotechnology enables us to synthesize large fragments of chromosomes and even entire genomes in a relatively short period of time, the cost required is also huge
The experimental techniques of Gibson assembly and Golden Gate assembly can meet some of our requirements for synthesizing small fragments of DNA into large fragments, but the standardized assembly elements and synthesis strategies similar to BioBrick in E. coli cannot be directly applied to eukaryotic microorganisms such as yeast

Method used

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  • Design and construction methods and application of standardized bio-elements
  • Design and construction methods and application of standardized bio-elements
  • Design and construction methods and application of standardized bio-elements

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] The construction of embodiment 1, HCkan-P, HCkan-O and HCkan-T

[0127] 1. Using pSMART HCKan as template, using SEQ ID No.1 and SEQ ID No.2 as primers, use ultra-fidelity DNA polymerase Q5 enzyme for PCR amplification (PCR program: 94°C for 3min; 94°C for 30s, 55°C 30s, 72°C for 40s, 30 cycles; 72°C for 7min; 4°C+∞), the PCR amplification product was obtained, the PCR amplification product was digested with BsaI, and the large vector fragment 1 was obtained. The sequence of the large vector fragment 1 is shown as SEQ ID Shown in No.3.

[0128] Replace the primers with SEQ ID No.4 and SEQ ID No.5, and the remaining experimental steps are the same as the above-mentioned experimental steps to obtain the PCR amplification product, digest the PCR amplification product with BsaI, and obtain the large vector fragment 2, the large vector fragment The sequence of 2 is shown in SEQID No.6.

[0129] Replace the primers with SEQ ID No.7 and SEQ ID No.8, and the remaining experim...

Embodiment 2

[0137] Embodiment 2, the construction of POT1-11

[0138] 1. Removal of BsaI and BsmBI sites on yeast vector pRS416

[0139] Using pRS416 as template, using F1 and R1 as primers, PCR amplification was performed using ultra-fidelity DNA polymerase Q5 enzyme (PCR program: 94°C for 3min; 94°C for 30s, 55°C for 30s, 72°C for 30s, 30 cycles; 72 ℃7min; 4℃+∞), a 2426bp PCR amplification product was obtained, and the PCR amplification product was digested with BsaI to obtain gene fragment A.

[0140] The primers were replaced with F2 and R2, and the remaining experimental steps were the same as the above-mentioned experimental steps to obtain a 1484bp PCR amplification product, which was digested with BsaI to obtain gene fragment B.

[0141]The primers were replaced with F3 and R3, and the remaining experimental steps were the same as the above-mentioned experimental steps to obtain a 1054bp PCR amplification product, which was digested with BsaI to obtain gene fragment C.

[0142] ...

Embodiment 3

[0161] Example 3, rapid assembly of β-carotene exogenous synthesis pathway in vitro

[0162] 1. The construction of each plasmid HCKan-P-TDH3, HCKan-P-ADH1, HCKan-P-TEF2 containing the promoter

[0163] (1) Acquisition of TDH3 promoter, ADH1 promoter and TEF2 promoter

[0164] Extract the genomic DNA of yeast BY4741, use it as a template, use F5 and R5 as primers, and use ultra-fidelity DNA polymerase Q5 enzyme to perform PCR amplification (PCR program: 94°C for 3min; 94°C for 30s, 55°C for 30s, 72°C 40s, 30 cycles; 72°C 7min; 4°C+∞), a 687bp PCR amplification product a1 was obtained, which contained a TDH3 promoter. The sequence of the TDH3 promoter is shown in SEQ ID No.42.

[0165] F5: 5'-agcgtgggtc tcgggcttca ttatcaatac tgccatttca aagaatacg-3';

[0166] R5: 5'-gtgctgggtc tcacatcttt gtttgtttat gtgtgtttat tcgaaact-3'.

[0167] The primers were replaced with F6 and R6, and the remaining experimental steps were the same as the above-mentioned experimental steps to obtain a ...

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Abstract

The invention discloses design and construction methods and application of standardized bio-elements. The construction method of the standardized bio-elements comprises the following steps that 1, a target genophore group, a promoter carrier group and a terminator carrier group are constructed. A series of standardized bio-elements are designed and constructed based on a yeast system, restriction enzymes II can be utilized for assembling the standardized bio-elements into an ultralong foreign DNA fragment, the foreign DNA fragment is a target product synthesis route composed of multiple genes, and the fragment is integrated to a specific site of a yeast chromosome through yeast homologous recombination for stable expression; a heterogenetic source in the target product yeast can be synthesized, optimum expression of the target product can be achieved by optimizing the promoters of all the genes, and therefore unique advantages and great significance are achieved in the optimization process of target product synthesis.

Description

technical field [0001] The invention relates to a design and construction method of a standardized biological element and its application; in particular to a design and construction method of a standardized biological element in Saccharomyces cerevisiae and its application, belonging to the fields of genetic engineering, metabolic engineering and synthetic biology. Background technique [0002] With the development of molecular biology and genetic engineering technology, the expression of exogenous metabolic pathways in Escherichia coli, yeast and even mammalian cells has been realized. Although these expression systems have been extensively studied, there are still many deficiencies. On the one hand, the metabolic pathway of a certain metabolite requires the synergy of multiple genes, and each gene often requires different degrees of expression regulation. For example, the nitrogen fixation pathway in Klebsiella oxytoca consists of 20 genes, and there are many unknown regu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N15/81C12N1/19C12P23/00C12R1/865
Inventor 戴俊彪林继伟吴庆余董俊凯
Owner TSINGHUA UNIV
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