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Genetic element, expression vector and application of genetic element and expression vector

A technology for expressing vectors and gene elements, which is applied in the field of synthetic biology and can solve problems such as unsatisfactory total production

Active Publication Date: 2016-10-12
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Under the action of RNA or DNA scaffolding, the yield of aliphatic hydrocarbons can be increased by 1.8 and 8.8 times respectively, but the total yield is still not satisfactory (only 27 or 44mg / L)

Method used

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  • Genetic element, expression vector and application of genetic element and expression vector
  • Genetic element, expression vector and application of genetic element and expression vector
  • Genetic element, expression vector and application of genetic element and expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Appropriate use of competitive AHR pathways to increase aliphatic hydrocarbon production.

[0049] Experimental Materials:

[0050] 1. Gene

[0051] 1) AAR: Acyl-ACP reductase, derived from Synechococcus elongatus PCC7942, protein sequence is accession number: YP_400611, codon optimized according to E. coli, the optimized DNA molecule encoding ADC7942 has the aar Nucleotide sequence, synthetic gene in GENEART.

[0052] 2) ADO: aldehyde group deformyl oxidase, derived from Synechococcus elongatus PCC7942, protein sequence is accession number: YP_400610, codon optimized according to Escherichia coli, the optimized DNA molecule encoding ADO has ado The nucleotide sequence shown is the gene synthesized in GENEART.

[0053] 3) Fatty alcohol oxidase (FAO), Candida tropicalis (ATCC 20336) protein information: GenBank: AAS46878.1, Accession: AY538780.1, GI: 44194456, according to E. coli for codons After optimization, the optimized DNA molecule encoding FAO has the nucleoti...

Embodiment 2

[0123] Example 2: Adaptation of fatty acid synthesis

[0124] Experimental Materials:

[0125] Strain information, reagents and culture medium are the same as in Example 1

[0126] experimental method:

[0127] 1. The construction method and plasmid information of YX33, YX10 and YX45 are the same as in Example 1.

[0128] 2. The plasmid information of each gene in the fatty acid synthesis pathway is shown in Table 5.

[0129] 3. Heat shock transformation of YX33, YX10 and YX45 with the plasmids in Table 5 into E.coli BL21(DE3) bacteria, and screen on LB solid plates. The cells were cultured in an incubator at 30°C. When YX33, YX10 and YX45 are co-transformed with Trc-Fab series genes into E.coli BL21(DE3) strain for fermentation, the IPTG concentration is divided into three concentrations of 1mM, 0.1mM and 0.01mM for induction.

[0130] 4. Ferment the strains transformed with each plasmid, and the method is the same as in Example 1.

[0131] 5. Extraction of the product, the method and p...

Embodiment 3

[0142] Example 3: Adaptation for lipid degradation

[0143] Experimental Materials:

[0144] The strain information, reagents and culture medium are the same as in Example 1.

[0145] experimental method:

[0146] 1. The construction method and plasmid information of YX33, YX10 and YX45 are the same as in Example 1.

[0147] 2. See Table 7 for lipid degradation series plasmid information.

[0148] 3. Heat shock transformation of YX33, YX10 and YX45 with the plasmids in Table 7 into E.coli BL21(DE3) bacteria, and screen on LB solid plates. The cells were cultured in an incubator at 30°C. When YX33, YX10 and YX45 are co-transformed with Trc-Fab series genes into E.coli BL21(DE3) strain for fermentation, the IPTG concentration is divided into three concentrations of 1mM, 0.1mM and 0.01mM for induction.

[0149] 4. Ferment the strains transformed with each plasmid, and the method is the same as in Example 1.

[0150] 5. Extraction of the product, the method and process are the same as in Exa...

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Abstract

The invention relates to the technical field of synthetic biology, in particular to a genetic element, an expression vector and application of the genetic element and the expression vector. The invention provides a novel method for improving the synthesis of fatty hydrocarbon microorganisms: the fatty hydrocarbon synthesis capability of escherichia coli is improved by dynamically balancing accumulation and conversion of a key intermediate metabolite, namely fatty aldehyde. In order to achieve this purpose, ADO is helped to balance the synthesis and conversion of the fatty aldehyde through a competition path by synergistically expressing a synthesis process of fatty alcohol; then, three modules for fatty acid synthesis, lipid degradation and electron transfer, which are related to dynamic balance of the fatty aldehyde, are matched with a fatty hydrocarbon synthesis module, and finally the artificial synthesis of high-yield fatty hydrocarbon from engineering escherichia coli is realized.

Description

Technical field [0001] The invention relates to the technical field of synthetic biology, in particular to gene elements, expression vectors and their applications. Background technique [0002] Compared with biological short-chain alcohol molecules, medium and long-chain hydrocarbons (C8-C18) have high calorific value (the highest heat generated per unit of fuel volume combustion), low vapor pressure (safe), and low moisture absorption (convenient storage and transportation) ) And other advantages, and are similar in structure and chemical characteristics to the current petroleum-based fuels, without mixing barriers, so it is a more ideal biological alternative fuel than short-chain alcohols. [0003] The synthesis of aliphatic hydrocarbons in microorganisms is mainly based on the extension of endogenous fatty acid carbon chains. In 2010, the research team led by Schirmer of LS9 company used the research idea of ​​genome mining to genetically compare 10 hydrocarbon-producing cyan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12P5/00
CPCC12N15/70C12N2800/101C12P5/00
Inventor 元英进曹英秀肖文海张金来丁明珠
Owner TIANJIN UNIV
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