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Design and construction methods of standardized biological components and their applications

A component and biological technology, applied in the direction of biochemical equipment and methods, microorganism-based methods, and other methods for inserting foreign genetic materials, can solve the problem of huge costs, can not be directly applied to eukaryotic microorganisms such as yeast, and is difficult to construct and optimize Exogenous metabolic pathways and other issues

Active Publication Date: 2020-04-03
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 of standardized biological components and their applications
  • Design and construction methods of standardized biological components and their applications
  • Design and construction methods of standardized biological components and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1. Construction of HCkan-P, HCkan-O and HCkan-T

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

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

[0129] Replace the primers with SEQ ID No.7 and SEQ ID No.8. The rest of the experimental steps are the same as the above experimental steps to obtain the PCR amplificatio...

Embodiment 2

[0137] Example 2. Construction of POT1-11

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

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

[0140] Replace the primers with F2 and R2, and the remaining experimental steps are the same as the above experimental steps to obtain a 1484bp PCR amplified product. BsaI digests the PCR amplified product to obtain gene fragment B.

[0141] Replace the primers with F3 and R3, and the remaining experimental steps are the same as the above experimental steps to obtain a 1054 bp PCR amplified product. BsaI digests the PCR amplified product to obtain gene fragment C.

[0142] The gene fragment A, the gene fragment B, and the gene frag...

Embodiment 3

[0161] Example 3. In vitro rapid assembly of β-carotene exogenous synthesis pathway

[0162] 1. The construction of each plasmid HCKan-P-TDH3, HCKan-P-ADH1, HCKan-P-TEF2 containing 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 for PCR amplification (PCR program: 94℃ 3min; 94℃ 30s, 55℃ 30s, 72℃ 40s, 30 cycles; 72°C 7min; 4°C+∞) to obtain a 687bp PCR amplification product a1, which contains the TDH3 promoter, and 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] Replace the primers with F6 and R6, and the remaining experimental steps are the same as the above experimental steps to obtain a 519 bp PCR amplification product b1, which contains the ADH1 p...

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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 method for designing and constructing a standardized biological element and its application; in particular to a method for designing and constructing a standardized biological element and its application in Saccharomyces cerevisiae, 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 E. coli, yeast and even mammalian cells has been achieved. Although these expression systems have been extensively studied, there are still many shortcomings. On the one hand, the metabolic pathway of a certain metabolite requires multi-gene synergy, and each gene often requires varying degrees of expression regulation. For example, the nitrogen fixation pathway in Klebsiella oxytoca consists of 20 genes, and there are many unknown regulatory factors, so ...

Claims

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Application Information

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Patent Type & Authority Patents(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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