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Intracellular scaffold, plasmids constructed by intracellular scaffold and application of plasmids

A technology of internal scaffolds and cells, which is applied in the direction of antibody mimics/scaffolds, microbial-based methods, and the use of vectors to introduce foreign genetic materials, etc. It can solve the problems of easy degradation, limited ligand protein types, scalability and stability bottlenecks, etc. Problems, to achieve the effect that is conducive to the aggregation of enzyme proteins and strong copolymerization

Active Publication Date: 2019-03-29
NAT UNIV OF DEFENSE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the application of RNA scaffolds is becoming more and more in-depth, due to the limited variety of ligand proteins and the fact that RNA is easier to degrade than DNA, its scalability and stability have always been its insurmountable bottleneck.

Method used

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  • Intracellular scaffold, plasmids constructed by intracellular scaffold and application of plasmids
  • Intracellular scaffold, plasmids constructed by intracellular scaffold and application of plasmids
  • Intracellular scaffold, plasmids constructed by intracellular scaffold and application of plasmids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] A TALE-DNA cell scaffold comprises DNA scaffold sequence SCAF1 and TALE-target molecule fusion protein.

[0081] The DNA scaffold sequence SCAF1 includes the binding sequence BM1, the binding sequence BM2 and the binding sequence BM3; wherein, BM1, BM2, and BM3 are concatenated according to the copy number of 1:1:1, and a 6bp length is designed between BM1, BM2, and BM3. interval sequence. BM1, BM2, and BM3 are arranged in a 1:1:1 manner to form a scaffold structure unit repeated four times.

[0082] BM1 (SEQ ID NO.1): 5'-ggaggcaccggtgg-3';

[0083] BM2 (SEQ ID NO.2): 5'-gataaacacctttc-3';

[0084] BM3 (SEQ ID NO. 3): 5'-gtcgatgccgagga-3'.

[0085] 6bp spacer sequence (SEQ ID NO.7): 5'-acagtt-3'

[0086] The DNA scaffold sequence SCAF1 is (SEQ ID NO.9):

[0087] ggaggcaccggtgg acagtt gataaacacctttc acagtt gtcgatgccgagga acagtt ggaggcac cggtgg acagttg ataaacacctttc acagtt gtcgatgccgagga acagtt ggaggcaccggtgg acagtt gata aacacctttc acagtt gtcgat...

Embodiment 2

[0102] A TALE-DNA cell scaffold, comprising DNA scaffold sequences SCAF1, SCAF2, SCAF3, SCAF4, SCAF5, SCAF6 and TALE1 / 2 / 3-target molecule 1 / 2 / 3 fusion protein.

[0103] The DNA scaffold sequence SCAF1 is consistent with Example 1.

[0104] The DNA scaffold sequence SCAF2, BM1, BM2 and BM3 were concatenated according to the copy number ratio of 1:1:1, and a spacer sequence with a length of 16 bp was designed between BM1, BM2 and BM3. BM1, BM2, and BM3 were arranged in a 1:1:1 manner to form a scaffold structure unit repeated four times.

[0105] The 16bp spacer sequence is: 5'-acagtagctacaacct-3' (SEQ ID NO.8).

[0106] The DNA scaffold sequence SCAF2 is (SEQ ID NO.10):

[0107] ggaggcaccggtgg acagtagctacaacct gataaacacctttc acagtagctacaacct gtcgatgc cgagga acagtagctacaacct ggaggcaccggtgga cagtagctacaacct gataaacacctttc acagtagctacaacct gtcgatgccgagga acagtagctacaacct ggaggcaccggtgg acagtagctacaacct gataaacacc tttc acagtagctacaacct gtcgatgccgagga acagtag...

Embodiment 3

[0129] A TALE-DNA cell scaffold, comprising DNA scaffold sequences SCAF1, SCAF2, SCAF3, SCAF4, SCAF5, SCAF6 and TALE1 / 2 / 3-target molecule A / B / C fusion protein. .

[0130] The DNA scaffold sequences SCAF1, SCAF2, SCAF3, SCAF4, SCAF5, SCAF6 are the same as in Example 1.

[0131] Among the TALE-target molecule fusion proteins, the target molecules are AtoB, HMGS, and HMGR; they are fused with TALE1, TALE2, and TALE3 to form TALE1-AtoB fusion protein, TALE2-HMGS fusion protein, and TALE3-HMGR fusion protein. The fusion method is consistent with Example 1.

[0132] TALE1-AtoB fusion protein specifically binds to the binding sequence BM1; TALE2-HMGS fusion protein specifically binds to the binding sequence BM2; TALE3-HMGR specifically binds to the binding sequence BM3.

[0133] 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 restriction endonu...

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Abstract

The invention discloses an intracellular scaffold, plasmids constructed by the intracellular scaffold and an application of the plasmids. The intracellular scaffold consists of a DNA scaffold sequenceand a TALE-target molecule fusion protein; the DNA scaffold sequence includes a binding sequence BM1, a binding sequence BM2 and a binding sequence BM3; the TALE-target molecule fusion protein includes a TALE1-target molecule fusion protein, a TALE2-target molecule fusion protein and a TALE3-target molecule fusion protein; the TALE1-target molecule fusion protein is bound to the binding sequenceBM1, the TALE2-target molecule fusion protein is bound to the binding sequence BM2, and the TALE3-target molecule fusion protein is bound to the binding sequence BM3. The DNA scaffold sequence is constructed on pSB1C3, BBa-J63009 or pSB4A5 plasmids, the TALE-target molecule fusion protein is constructed on pET28a (+) plasmids, and the obtained transformed plasmids are co-transformed into escherichia coli, IPTG-induced expression is performed, and the obtained plasmids can be used for improving the yield of mevalonic acid in mevalonic acid synthesis reaction.

Description

technical field [0001] The present invention relates to the fields of bioengineering and biomanufacturing, in particular to the invention of a new type of intracellular scaffold structure by using molecular biology technology and gene editing technology, the plasmid constructed by it and the application of the plasmid. Background technique [0002] Terpenoids are a class of highly diverse natural products, usually isolated from plants, microorganisms and marine organisms, and can be used as fragrances, essential oils, health supplements, medicines, etc. For example, paclitaxel and artemisinin, terpene compounds extracted from plants, are used as anticancer and antimalarial drugs, respectively. However, the natural production of these compounds is very small, and only a few target products can be obtained by purification from biological products. At the same time, because they are mixed with a large number of other natural products, their purity is very low, and they are eas...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/70C12P7/42C12R1/19
CPCC07K14/00C07K2319/00C12P7/42
Inventor 朱律韵谢斯思朱凌云邱鑫源朱楚姝吴小敏张东裔
Owner NAT UNIV OF DEFENSE TECH
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