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Phage-assisted cellooligosaccharide transporter continuous directed evolution system and method

A directed evolution, cello-oligosaccharide technology, applied in the field of genetic engineering, can solve the problems of reducing evolutionary efficiency, human intervention, and high cost, and achieve the effects of avoiding human intervention, high evolutionary efficiency, and improving transport capacity.

Pending Publication Date: 2022-07-29
GUANGZHOU INST OF ADVANCED TECH CHINESE ACAD OF SCI
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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.) and endow these proteins with new properties, but the PACE evolution system needs to rely on expensive operating equipment, so the cost is high, and this system is not suitable for the directed evolution of cellooligosaccharide transporters
Our previous patent "A Plasmid and Phage-Assisted Continuous Directed Evolution System and Directed Evolution Method" improved the PACE evolution system and successfully used it for the evolution of cellooligosaccharide transporters, but it still has certain defects. First, there are many human interventions; second, the induction method to increase the mutation rate is arabinose induction, which may interfere with the PACE system induced by its own cellooligosaccharides
Third, the local background expression of PIII has a negative impact on the evolutionary system and will reduce the evolutionary efficiency

Method used

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  • Phage-assisted cellooligosaccharide transporter continuous directed evolution system and method
  • Phage-assisted cellooligosaccharide transporter continuous directed evolution system and method
  • Phage-assisted cellooligosaccharide transporter continuous directed evolution system and method

Examples

Experimental program
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Embodiment 1

[0033] This embodiment provides a phage-assisted continuous directed evolution system of cello-oligosaccharide transporter, including: AV1 plasmid, AV2 plasmid and phage, the nucleic acid sequences of AV1 plasmid and AV2 plasmid are respectively SEQ ID NO.1 and SEQ ID NO.2 shown. Phage, that is, the phage SP-LacY in which the M13 phage gene gIII is replaced by the target gene LacY: PCR amplification of the M13 phage macroframe SP (without the gIII gene), PCR amplification of the wild-type lacY gene, and then using gibson to connect the M13 phage macroframe The SP and lacY genes form the SP-lacY double-stranded plasmid, and the gene map is as follows figure 1 As shown, the nucleic acid sequence is shown in SEQ ID NO.3.

[0034] The method for obtaining wild-type SP-LacY is as follows:

[0035]1) AV1 and AV2 co-transformed FM15 competent cells to obtain the host FM15-AV1 / AV2, which expresses on AV1 before cellobiose is transported into the cell without LacY protein (amino acid...

Embodiment 2

[0043] This example investigates the substrate specificity of LacY using the evolutionary system of Example 1, and adopts cellobiose, and the specific operations are as follows:

[0044] The initial phage was wild-type SP-lacY, titer 1×10 9 pfu / mL, the host is FM15-AV1 / AV2 (OD600=0.4), 1-2 uL of host bacteria are spotted in the center of LB soft agar (containing 1 mM cellobiose), and three spots are placed about 1 cm away from the host site. Phage droplets (1-2uL), three large phage fan-shaped spots can be seen the next day; the second round is similar, but the cellobiose concentration is reduced to 200uM, and the phage is diluted 100 times with the phage after the first round of evolution. board; for the following rounds, the third round is 40uM, the fourth round is 10uM, and the fifth round is 2uM. Until the formation of fan-shaped spots is difficult or new mutations no longer occur. Each round of sampling and sequencing was used to detect the accumulation of LacY mutation...

Embodiment 3

[0047] Evolution product LacY A177V Validation of transport activity using cellobiose:

[0048] Construction of plasmid AV3-LacY wild and AV3-LacY A177V , which carry wild-type LacY or mutant LacY, respectively A177V , and both are constitutively expressed by promoter J23109 (AV3-LacY wild The plasmid map of Figure 7 shown, AV3-LacY wild Sequence structure such as SEQ ID NO.7); construct plasmid AV4 (plasmid map such as Figure 8 shown, SEQ ID NO. 8), which carries the reporter gene GFP, is initiated by pTAC-CelRS, and is controlled bi-levelly by CelR and the hairpin.

[0049] Plasmid AV3-LacY wild and AV3-LacY A177V Strain S1030-AV4 was transformed respectively to obtain engineering strain S1030-AV4 / AV3-LacY wild and S1030-AV4 / AV3-LacY A177V .

[0050] S1030-AV4 / AV3-LacY wild and S1030-AV4 / AV3-LacY A177V The cells were cultured in LB medium to log phase, 10uM cellobiose was added to induce GFP expression for 3h, centrifuged and washed twice with PBSbuffer, and t...

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Abstract

The invention provides a bacteriophage-assisted cellooligosaccharide transporter continuous directed evolution system and a bacteriophage-assisted cellooligosaccharide transporter continuous directed evolution method. The directed evolution system comprises AV1 plasmid, AV2 plasmid and bacteriophage, and nucleotide sequences of the AV1 plasmid and the AV2 plasmid are respectively shown as SEQ ID NO.7 and SEQ ID NO.8. The invention further provides a preparation method of the bacteriophage-assisted cellooligosaccharide transporter continuous directed evolution system. The method for directed evolution of the cello-oligosaccharide transporter comprises the following steps: co-transforming AV1 plasmid and AV2 plasmid into host bacteria to obtain engineering bacteria for evolution; the phage and the engineering bacteria for evolution obtained in the step 1 are subjected to multiple rounds of evolution culture in a culture medium containing the cellooligosaccharide, in the multiple rounds of evolution culture process, the concentration of the cellooligosaccharide in the culture medium is decreased progressively so as to increase the screening pressure, and the mutant protein is obtained. The invention designs a visual bacteriophage-assisted continuous directed evolution system, the system comprises AV1 plasmid and AV2 plasmid, the visual high-throughput evolution process of the cello-oligosaccharide transporter can be realized by adopting the evolution system to evolve the cello-oligosaccharide transporter, excessive human intervention is avoided, the evolution efficiency is higher and more sensitive, and the method is suitable for large-scale popularization and application. Further, the transport capacity of the LacY on the cellooligosaccharides is greatly improved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a phage-assisted continuous directed evolution system and method of cellooligosaccharide transporter. Background technique [0002] In the genome, about 30% of the gene products are membrane proteins, this ratio shows the importance of membrane proteins in the organism. Membrane proteins mainly include signal receptors, transport proteins, ion channel proteins and some enzymes, which are essential for 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 of the instability and insolubility of membrane proteins, it is difficult for researchers to obtain high purity The three-dimensional structure of membrane proteins was analyze...

Claims

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Application Information

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IPC IPC(8): C12N15/70C07K14/245C12N15/31C12N1/21C12R1/19
CPCC12N15/70C07K14/245C12N2795/14143
Inventor 李小明崔金明刘陈立
Owner GUANGZHOU INST OF ADVANCED TECH CHINESE ACAD OF SCI
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