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A phage-assisted continuous directed evolution system and method for multiple bacteria

A directed evolution and phage technology, applied in biochemical equipment and methods, viruses/phages, bacteria, etc., can solve the problems of mutual interference of different genetic elements and low efficiency of phage proliferation and evolution

Active Publication Date: 2021-04-20
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0006] In order to solve the problem of low efficiency of phage proliferation and evolution in the phage-assisted evolution system and the mutual interference of different genetic elements recognized by phage SM (or the target gene carried by SM) before and after evolution, the present invention provides a phage-assisted continuous directed evolution of multiple bacteria systems and methods

Method used

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  • A phage-assisted continuous directed evolution system and method for multiple bacteria
  • A phage-assisted continuous directed evolution system and method for multiple bacteria
  • A phage-assisted continuous directed evolution system and method for multiple bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068]Example 1 Evolution desired host fungi preparation

[0069]1) The host fungi S1 carrying HP1 and IP plasmids, in LB medium containing 50 μg / ml of carboxylcin and 25 μg / ml chloramphenicol-resistant, to OD600 = 0.6 at 37 ° C, 220 rpm. The bacterial liquid is placed at 4 ° C for spare, and the preservation time is no more than 1 day.

[0070]2) The host bacteria S2 carrying HP2 and IP plasmids was cultured to OD600 = 0.6 at 37 ° C, 220 rpm in LB medium containing 50 μg / ml of carboenomycin and 25 μg / ml chloramphenicol. The bacterial liquid is placed at 4 ° C for spare, and the preservation time is no more than 1 day.

[0071]3) The host fungi S3 carrying HP2, HP3, and IP plasmids, in LB medium containing 50 μg / ml of carboenomycin, 50 μg / ml of magnifier and 25 μg / ml chloramphenicol resistance, at 37 ° C, 220 rpm Conditions were cultured to OD600 = 0.6.12000 rpm 1min centrifugal collected bacteria, and then resuspended in an equal volume of 50 μg / ml of carboenomycin and 25 μg...

Embodiment 2

[0077]Example 2S5 SMP observation method

[0078]1) In a 10 cm bacterial culture plate, a layer of 10 ml of 2% agarose gel was paved, and 20 min was placed at room temperature to solidify.

[0079]2) When the SM concentration is unknown, it is necessary to press SM to press 0 times and 10.1，2，3，4，5The proportion of times is diluted in a continuous gradient.

[0080]3) Take 6 groups of 200 μl of Example 1, S5 host bacteria prepared, each group added to SM 10 μl of different dilution gradients in "2), and then 4 mL preserved at 55 ° C containing 0.4% agar lb medium and final The concentration of 50 μg / ml carboxylmycin. After mixing the vortex shock mixer, the sample is placed into the plate prepared in "1)".

[0081]4) Culture overnight in a biochemical incubator at 37 ° C.

[0082]5) Observe the number of plazots formed by the statistical gradient dilution sample, and the SM concentration is calculated.

Embodiment 3

[0083]Example 3S6 and SM plaque observation method in S7 host bacteria

[0084]Similarly to Example 2, the S6 bacterium formed in Example 1 was replaced with the S6 bacterium, which was prepared in Example 1, which was replaced with the second embodiment.

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Abstract

The invention discloses a phage-assisted multi-bacteria continuous directed evolution system and method. The system includes phage SM carrying a target gene to be evolved, various auxiliary plasmids HP supporting the proliferation of different SMs before and after evolution, and a mutation-inducing plasmid IP. Different HPs are placed in different host bacteria, which can effectively improve the proliferation and evolution efficiency of phages, and at the same time avoid the mutual interference between different gene elements, which has good practicability and can be used for the directed evolution of multiple genes.

Description

Technical field[0001]The present invention relates to the field of biological evolution, and more particularly to a phage-assisted multi-bacterial continuous orientation evolutionary system and method.Background technique[0002]Directional evolution (also known as laboratory evolution) is a powerful technical approach that produces biomolecules with a designated function by driving a biological evolution process. The generated biomolecules have been widely used in industrial production, bioengineering, medical development and many other areas. Harvard University David R Liu laboratory has developed a continuous evolutionary system based on phage growth (phage auxiliary evolution, PACE). It mainly includes three modules of SM, HP, IP: Phage Module (SM): Phage M13 gene packaging and the GIII gene needed to invade the Hydrocarbatus is removed, inserted into the genes of the biomolecule to be carried out, and the phage lacks GIII, It is impossible to invade a host to generate a child; mu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/73C12N1/21
CPCC12N1/20C12N15/73C12N2800/101
Inventor 刘陈立赖旺生魏婷赵国屏
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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