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Gene elements, expression vectors and their applications

A technology for expression vectors and genes, applied in the field of synthetic biology, which can solve the problems of unsatisfactory total output and other problems

Active Publication Date: 2019-12-31
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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  • Gene elements, expression vectors and their applications
  • Gene elements, expression vectors and their applications
  • Gene elements, expression vectors and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Appropriate utilization of competing AHR pathways to enhance aliphatic hydrocarbon production.

[0049] Experimental Materials:

[0050] 1. Gene

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

[0052] 2) ADO: aldehyde deformyl oxidase, derived from Synechococcus elongatus PCC7942, the protein sequence is accession number: YP_400610, the codon is 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 (fatty alcohol oxidase, FAO), Candida tropicalis (Candidatropicalis, ATCC 20336) protein information is: GenBank: AAS46878.1, Accession: AY538780.1, GI: 44194456, acc...

Embodiment 2

[0123] Example 2: Adaptation of fatty acid synthesis

[0124] Experimental Materials:

[0125] Bacterial strain information, reagent and substratum etc. are identical with embodiment 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. See Table 5 for the plasmid information of each gene in the overexpression fatty acid synthesis pathway.

[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. Cells were cultured in a 30°C incubator. When YX33, YX10 and YX45 were co-transformed with Trc-Fab series genes into E.coli BL21(DE3) strain for fermentation, the IPTG concentration was divided into three concentrations of 1mM, 0.1mM and 0.01mM for induction.

[0130] 4. Fermenting the bacterial strain transformed with each plasmid, the process is the same as in Example 1.

[0131] 5....

Embodiment 3

[0142] Example 3: Adaptation for lipid degradation

[0143] Experimental Materials:

[0144] The strain information, reagents and medium etc. 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 the plasmid information of lipid degradation series.

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

[0149] 4. Fermenting the bacterial strain transformed with each plasmid, the process is the same as in Example 1.

[0150] 5. The extraction of product, method process is ...

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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 applications thereof. Background technique [0002] Compared with biological short-chain alcohol molecules, medium- and long-chain hydrocarbons (C8-C18) have high calorific value (highest heat generated by combustion per unit fuel volume), low vapor pressure (safety), and low hygroscopicity (convenient for storage and transportation) ) and other advantages, and are similar to the current petroleum-based fuels in structure and chemical properties, and there is no mixing barrier, so it is a more ideal bio-alternative fuel than short-chain alcohols. [0003] The synthesis of aliphatic hydrocarbons in microorganisms is mainly based on the elongation 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 compare the genes of 10 hydrocarbon-producing ...

Claims

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Application Information

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Patent Type & Authority Patents(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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