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Standardized, high-accuracy and general functional module construction method

A functional module, high-precision technology, applied in the field of synthetic biology, can solve problems that do not involve the assembly of multiple modules

Active Publication Date: 2015-03-18
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The Standardized Assembly of Transcriptional Units method uses Golden Gate enzymes to construct a single yeast expression cassette, and currently does not involve the assembly of multiple modules

Method used

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  • Standardized, high-accuracy and general functional module construction method
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  • Standardized, high-accuracy and general functional module construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of pLD-Blunt plasmid vector, pLD-Blunt plasmid vector is shown by SEQ ID NO.28.

[0044] In the present invention, pLD-Blunt is the only cloning vector artificially constructed for placing genetic elements, and its construction process is as follows:

[0045](1) Select the pEASY-Blunt kit of full gold, take out 1 μL of pEASY-Blunt reagent, add 4 μL of water, react at 25°C for 5 minutes, transform Escherichia coli, pre-coat IPTG and X-gal on the plate, and use blue-white screening to obtain blue colonies.

[0046] (2) Insert the blue colony into LB-Amp liquid medium, culture overnight at 37°C, and extract the pEASY-Blunt empty plasmid vector.

[0047] (3) Use the primer pLD-Blunt1-F shown in SEQ ID NO.19 and the primer pLD-Blunt1-R shown in SEQ ID NO.20 as the upstream primer and downstream primer, and use the pEASY-Blunt empty plasmid vector as a template, The BsaI point mutation was introduced into the Amp resistance gene, and the main part of ...

Embodiment 2

[0052] Example 2 Construction of pRS425K plasmid vector, the sequence of pRS425K is shown in SEQ ID NO.29.

[0053] In the present invention, pRS425K is an artificially constructed cloning vector for placing the module tool sequence, and its construction process is as follows:

[0054] (1) Select the pEASY-Blunt kit of full gold, take out 1 μL of pEASY-Blunt reagent, add 4 μL of water, react at 25°C for 5 minutes, transform Escherichia coli, pre-coat IPTG and X-gal on the plate, and use blue-white screening to obtain blue colonies.

[0055] (2) Insert the blue colony into LB-Amp liquid medium, culture overnight at 37°C, and extract the plasmid, which is the pEASY-Blunt empty plasmid vector. At the same time, the Escherichia coli strain containing pRS425 was inoculated to extract the pRS425 empty plasmid vector.

[0056] (3) Using the primer pRS425K1-F shown in SEQ ID NO.23 and the primer pRS425K1-R shown in SEQ ID NO.24 as the upstream primer and downstream primer, and using...

Embodiment 3

[0061] Example 3 Constructing the functional module of the green precursor deoxychromoviridans for the synthesis of violacein

[0062] Violacein is a bacteriostatic compound derived from Chromobacterium violaceum. Violacein is synthesized by cells using tryptophan as a substrate through five enzyme reactions: VioA, VioB, VioE, VioD, and VioC. After only the first three steps, the cells synthesize a green product called deoxychromoviridans, which makes yeast colonies appear green.

[0063] Therefore, in this example, three strength functional modules were designed for the genes of the three enzymes VioA, VioB, and VioE. On this basis, different functional modules were used to co-transform yeast, and different modules were combined to obtain combined functional modules. Enables Saccharomyces cerevisiae to produce deoxychromoviridans, making colonies green.

[0064] In this example, VioA is represented by SEQ ID NO.1, VioB is represented by SEQ ID NO.2, and VioE is represented ...

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Abstract

The invention discloses a standardized, high-accuracy and general functional module construction method, wherein the construction method comprises the following steps: (1) adding a standardized site to a source gene to form a genetic element, linking the genetic element into a plasmid pLD-Blunt to form a plasmid with the genetic element, and carrying out sequence verification; (2) taking the genetic element from the plasmid with the genetic element, and linking to a following notch in a module tool plasmid: terminator 1-promoter-AATG-notch-TAAA-terminator 2; and constructing plasmids with different functional modules; and (3) taking different functional modules from the plasmids with the different functional modules, and co-transforming with a yeast plasmid vector subjected to restriction enzyme digestion into saccharomyces cerevisiae, constructing the standardized, high-accuracy and general functional modules, and storing in the plasmids. The construction method has the advantages of universality, standardization, high accuracy, and free selection.

Description

technical field [0001] The invention relates to the field of synthetic biology, in particular to a method for constructing functional modules. Background technique [0002] Functional modules play a crucial role in synthetic biology research. Artificial optimization and reconstruction of endogenous metabolic pathways, transformation of chassis cell genomes, rapid assembly of multiple exogenous genetic elements, and artificial design and construction of new life functions all rely on functional modules. Therefore, designing a set of standardized, high-precision, and general-purpose functional module construction and assembly methods, so that it can quickly and efficiently obtain the required modules, has become an urgent bottleneck technology for synthetic biology. [0003] The ideal construction method of functional modules urgently needed by synthetic biology should have the following salient features: 1) For any genetic element, no site pre-modification is required. 2) D...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/63C12N1/19
Inventor 元英进刘夺李炳志王霞申明华胡梦龙杜昊星苏皖宋田青翟芳郭雪娇郭睿
Owner TIANJIN UNIV
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