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Intracellular scaffold structure and method

A scaffold structure and inner scaffold technology, applied in the field of intracellular scaffold structure, can solve problems such as stability and scalability limitations

Inactive Publication Date: 2016-10-12
NAT UNIV OF DEFENSE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is still based on the combination of guide RNA (gRNA) and ligand protein, which belongs to the category of RNA scaffold, and its stability and scalability are limited.

Method used

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  • Intracellular scaffold structure and method
  • Intracellular scaffold structure and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction method of TALE-DNA scaffold system prokaryotic expression plasmid

[0043] The TALE expression sequence was constructed using Golden Gate TALEN and TAL Effector Kit 2.0, and the DNA scaffold sequence was synthesized by DNA synthesis. Utilizing the properties of the restriction endonuclease Spe1 and Xba1 homologous enzymes, a multiple cloning site system of EcoR1, Xba1, Spe1 and Pst1 was designed on the pSB1C3 plasmid, and the Plac promoter, Plac promoter, and Nucleic acid elements such as ribosome binding site (RBS), TALE, split GFP, tryptophan monooxygenase (IAAM), indole acetamide hydrolase (IAAH), terminator (Ter) or DNA scaffold sequence (Scaffold) . Specifically construct the following plasmids: construct pSB1C3-Plac-RBS-TALE1-GFP1-Ter-Scaffold1 (pT1-GFP1-S1), pSB1C3-Plac-RBS-TALE2-GFP2-Ter-Scaffold2 (pT2-GFP2-S2) and pSB1C3- The Plac-RBS-TALE3-GFP2-Ter-Scaffold1 (pT3-GFP2-S1) plasmid was used to detect the binding ability of the TALE-GFP f...

Embodiment 2

[0044] Example 2 Verification of the binding ability of the TALE-GFP fusion protein provided by the present invention to the DNA scaffold

[0045] ChIP-PCR was used to test whether the fusion protein of TALE and heterologously expressed protein (GFP1 / 2) could effectively bind to the plasmid DNA scaffold. Firstly, the constructed pSB1C3-1, pSB1C3-2 and pSB1C3-3 plasmids were respectively transformed into Escherichia coli BL21 (DE3) strain competent, positive clones were screened, and inoculated in 5 mL of LB liquid medium containing ampicillin (50 μg / ml) , placed on a shaker at 37°C, and cultivated at 250rpm for 5 hours, then added the culture to LB liquid medium containing ampicillin (50 μg / ml) at a ratio of 1:100, placed on a shaker at 37°C, and continued to cultivate at 250rpm. Determination of bacterial growth curve, wait for OD 600 When the temperature reaches 0.6-1.0, add IPTG to make the final concentration 1mM, and then transfer to 20°C, 25°C or 30°C incubator at 200rp...

Embodiment 3

[0047] Example 3 Verification of the colocalization effect of the TALE-DNA scaffold system provided by the present invention on heterologous proteins

[0048] The colocalization effect of the TALE-DNA scaffold system provided by the present invention on heterologous proteins was verified by splitting GFP experiments. The five groups of plasmids pT1-G1 / T3-G2-S1, pT1-G1 / T2-G2-S2, pT1-G1 / T2-G2-S3, pT1-G1 / T3-G2, pT1-G1 / T2-G2 were respectively Transform Escherichia coli BL21 (DE3) strain to be competent, and after expanding the culture of positive clones, induce overnight at 25° C. with 1 mM IPTG. The group without IPTG was used as the uninduced control group. Bacterial samples were subsequently collected, and the OD of each group was measured with a fluorescent microplate reader Fluoroskan Ascent FL (Thermo Scientific Company) 600 and fluorescence intensity FI (excitation light wavelength: 488nm; emission light wavelength: 538nm). Such as Figure 4 As shown in B, FI / OD of pT1-...

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Abstract

The invention discloses a novel intracellular scaffold structure based on TALE (transcription activator-like effectors) and a method. The intracellular scaffold structure comprises a DNA scaffold sequence constructed on a plasmid vector and two sections of adjacent TALE-enzyme infusion protein binding sequences. By virtue of a TALE-DNA scaffold technology, an intracellular heterogeneous metabolic pathway enzyme system is co-regionalized, so that a metabolism rate is improved and the yield of target metabolites is increased.

Description

technical field [0001] The present invention relates to the fields of bioengineering and biomanufacturing, in particular to inventing a novel intracellular scaffold structure using molecular biology technology and gene editing technology, as a new method to increase the yield of target products in engineered microorganisms. Background technique [0002] Naturally occurring metabolic processes in eukaryotic cells can be concentrated in various subcellular compartments constructed by the endomembrane system, compartmentalizing the enzymatic reaction system. For example, the citric acid cycle and β-oxidation of fatty acids are concentrated in the mitochondria, while protein processing and modification take place in the Golgi apparatus. The regionalization of enzymes is conducive to the mutual connection and close cooperation of enzymes in the same metabolic pathway, and the high concentration of enzymes, coenzymes and substrates, thereby increasing the rate of enzymatic reactio...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/55C12N15/66
CPCC12N9/22C12N15/63C12N15/66
Inventor 朱律韵朱凌云邱鑫源吴小敏章媛范冬雨朱楚姝张东裔
Owner NAT UNIV OF DEFENSE TECH
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