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Phage-assisted multi-bacterial continuous directed evolution system and method

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, and achieve the effect of improving evolution efficiency

Active Publication Date: 2017-12-01
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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  • Phage-assisted multi-bacterial continuous directed evolution system and method
  • Phage-assisted multi-bacterial continuous directed evolution system and method
  • Phage-assisted multi-bacterial continuous directed evolution system and method

Examples

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Effect test

Embodiment 1

[0068] The preparation of the host bacteria required for the evolution of embodiment 1

[0069] 1) The host strain S1 carrying the HP1 and IP plasmids was cultured in LB medium containing 50 μg / ml carbenicillin and 25 μg / ml chloramphenicol resistance at 37° C. and 220 rpm until OD600 = 0.6. Store the bacterial solution at 4°C for later use, and the storage time should not exceed 1 day.

[0070] 2) The host strain S2 carrying the HP2 and IP plasmids was cultured in LB medium containing 50 μg / ml carbenicillin and 25 μg / ml chloramphenicol resistance at 37° C. and 220 rpm until OD600=0.6. Store the bacterial solution at 4°C for later use, and the storage time should not exceed 1 day.

[0071] 3) The host bacteria S3 carrying the HP2, HP3 and IP plasmids were placed in the LB medium containing 50 μg / ml carbenicillin, 50 μg / ml spectinomycin and 25 μg / ml chloramphenicol resistance, at 37 ° C, 220 rpm Cultivate to OD600=0.6. Centrifuge at 12000 rpm for 1 min to collect the bacteria,...

Embodiment 2

[0077] SM phage plaque observation method in embodiment 2S5 host bacterium

[0078] 1) Spread a layer of 10ml of 2% agarose gel on a 10cm bacterial culture plate, and let it solidify at room temperature for 20min.

[0079] 2) When the concentration of SM is unknown, it is necessary to divide SM by 0 times and 10 1，2，3，4，5 Doubling serial dilutions were performed.

[0080]3) Take 6 groups of 200 μL of the S5 host bacteria prepared in Example 1, add 10 μL of SM with different dilution gradients in “2)” to each group, and then add 4 ml of LB medium containing 0.4% agar stored at 55° C. and the final Concentration 50 μg / ml carbenicillin. After mixing on a vortex mixer, the samples were spread onto the plates prepared in "1)".

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

[0082] 5) Observing and counting the number of plaques formed by each serially diluted sample, and calculating the SM concentration.

Embodiment 3

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

[0084] Same as in Example 2, it is sufficient to replace the host bacteria forming plaques with the S6 bacteria and S7 bacteria prepared in Example 1, respectively.

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Abstract

The invention discloses phage-assisted multi-bacterial continuous directed evolution system and method. The system includes: a phage SM which carries an evolution target gene, a plurality of assistant plasmids HP which support different SM proliferations before and after the evolution, and a plasmid IP which induces mutation. In the system, the different HPs are arranged in different host bacteria, so that proliferation and evolution efficiencies of the phage are effectively improved, and meanwhile, mutual interference between different gene components is avoided. The system and method have great practicability and can be applied to directed evolution of various genes.

Description

technical field [0001] The invention relates to the field of biological evolution, in particular to a phage-assisted multi-bacteria continuous directed evolution system and method. Background technique [0002] Directed evolution (also known as laboratory evolution) is a powerful technical approach that can be used to generate biomolecules with specified functions by harnessing the process of biological evolution. The biomolecules produced have been widely used in many fields such as industrial production, bioengineering, and pharmaceutical development. A continuous evolution system based on phage growth (phage-assisted continuous evolution, PACE) was developed in the laboratory of David R Liu at Harvard University. It mainly includes three modules: SM, HP, and IP: Phage module (SM): the gIII gene required for packaging and infecting host bacteria in the phage M13 gene is excised, and inserted into the gene of the biomolecule to be evolved, where the phage lacks gIII, Unab...

Claims

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Application Information

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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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