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Plasmid, phage-assisted continuous directed evolution system and directed evolution method

A directed evolution, bacteriophage technology, applied in biochemical equipment and methods, microorganism-based methods, bacteria, etc., can solve problems such as being unsuitable for directed evolution of membrane proteins and not involving the field of membrane proteins.

Active Publication Date: 2019-06-28
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

David R Liu's team successfully evolved T7 RNA polymerase (NATURE.2011April; 472:498–505), protease (Nature Communications, 2014, 5:5352.), DNA binding protein (Nature Methods, 2015, 12 (10 ):939.) etc. and endowed these proteins with new properties, but did not involve the field of membrane proteins, the existing PACE system is not suitable for directed evolution of membrane proteins

Method used

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  • Plasmid, phage-assisted continuous directed evolution system and directed evolution method
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  • Plasmid, phage-assisted continuous directed evolution system and directed evolution method

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Packaging of bacteriophage SP-lacY carrying quasi-evolved genes

[0056] 1) Construction of the gIII protein expression plasmid AP1: the gIII protein is promoted by the phage shock promoter J23109, and the nucleic acid sequence (SEQ ID NO.3) of the recognition site of the CelR protein is inserted downstream of the promoter. AP1 map see figure 2 .

[0057] 2) Construction of expression plasmid AP2 of functional protein CelR protein: CelR is expressed by a constitutive promoter ( image 3 ).

[0058] 3) The mutagenic plasmid MP was donated by David R Liu's laboratory, see the map Figure 4 .

[0059] 4) AP1, AP2 and MP co-transform S1030 competent cells to obtain host S1030-AP1 / AP2 / MP. Before the host does not have LacY protein to transport cellobiose into the cell, the CelR protein expressed on AP2 and the CelR protein on AP1 The CelR recognition site binds to repress the expression of the downstream gene gIII; MP is a plasmid that improves mutations and is induced...

Embodiment 2

[0065] Verification of the relationship between the concentration of cellobiose and the propagation speed of bacteriophage SP-lacY

[0066] 1) The host S1030-AP1 / AP2 / MP LB medium was cultured to the logarithmic phase (OD 600 = 0.4).

[0067] 2) Dilute the wild-type SP-lacY phage and add it to the above logarithmic phase host to make the initial concentration in the system 50pfu / mL.

[0068] 3) The final concentration gradient of cellobiose is 29mM, 14.5mM, 2.9mM, 1.45mM, 0.29mM, 0.0029mM, 0.00029mM, 0.00mM.

[0069] 4) Take samples every 15 minutes for each cellobiose gradient, and detect the proliferation of phage SP-lacY in the system. The results are as follows: Image 6 shown.

[0070] Image 6 In , the concentration of cellobiose from 0mM to 0.029mM basically coincides with the abscissa.

[0071] Image 6 The experimental results show that the proliferation rate of phage is directly proportional to the concentration of cellobiose, and the reduction of the concentrat...

Embodiment 3

[0073] Substrate specificity of PACE evolved LacY

[0074] 1) The host S1030-AP1 / AP2 / MP was cultured to the logarithmic phase (OD 600 = 0.4).

[0075] 2) The first round of evolution, in the 1mL evolution system, the initial cellobiose final concentration was 29mM, and the initial wild-type SP-lacY phage was 1.2×10 5 pfu / mL, the final concentration of arabinose is always 1%, add host S1030-AP1 / AP2 / MP to 1mL, 37°C, 150RPM for 1h. Sampling, serial dilution, mixed 10 μL of diluted phage with 190 μL logarithmic phase host S1030-AP1 / AP2 / MP, placed at 37°C for 15 minutes, mixed with 1mL of 0.5% soft agar containing 0.5% cellobiose at 50°C, and evenly Spread on a solid agar plate with a diameter of 60 cm, let it stand for 10 minutes, and culture it overnight at 37°C after solidification, calculate the number of phage plaques, and determine the concentration of phage in the system. Take a single plaque, use primers SP1-F and SP1-R to amplify the LacY gene fragment on the phage, and...

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Abstract

The invention relates to the field of directed evolution of membrane proteins, in particular to a plasmid, a phage-assisted continuous directed evolution system and a directed evolution method. The plasmid comprises one or two of a plasmid AP1and a plasmid AP2; the plasmid AP1 carries a gIII gene, and a functional protein recognition site is arranged between a promoter of the plasmid and a ribosome binding site; the plasmid AP2 carries a functional protein gene. According to the plasmid, the phage-assisted continuous directed evolution system and the directed evolution method, the new assisting plasmids AP1 and AP2 are designed, the transport activity of the target protein to intracellular and extracellular substrate molecules and the gIII expression on the AP1 are coupled, the reproductive capability of SP and the activity of the target protein are coupled in the indirect mode, and the effect of continuous directed evolution of the target protein is achieved.

Description

technical field [0001] The invention relates to the field of membrane protein directed evolution, in particular to a plasmid and phage-assisted continuous directed evolution system and directed evolution method. Background technique [0002] In the genome, about 30% of the gene products are membrane proteins, which shows the importance of membrane proteins in organisms. Membrane proteins mainly include signal receptors, transporters, ion channel proteins and some enzymes, which are crucial to cell metabolism, physiological balance and intracellular regulation. In the process of drug development and design, many membrane proteins are the targets of drug design. However, the lack of membrane protein structure and biochemical information restricts the development of drug design, because the instability and insolubility of membrane proteins make it difficult for researchers to obtain high-purity Analyze and study the three-dimensional structure of the membrane protein. For the...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N1/21C12R1/19
Inventor 刘陈立李小明崔金明
Owner SHENZHEN INST OF ADVANCED TECH
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