A method for constructing a bidirectional screening system for directed evolution of lead-binding proteins

A technology for binding proteins and lead ions, which can be used in chemical instruments and methods, biochemical equipment and methods, and botanical equipment and methods, and can solve problems such as limited application potential.

Active Publication Date: 2022-03-29
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, although the lead-binding protein PbrR can selectively recognize lead ions as a metal-responsive element, it also has a certain binding ability to non-target metal ions such as zinc ions, which limits the application potential of this metal regulatory protein for the specific detection of metal ions.

Method used

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  • A method for constructing a bidirectional screening system for directed evolution of lead-binding proteins
  • A method for constructing a bidirectional screening system for directed evolution of lead-binding proteins
  • A method for constructing a bidirectional screening system for directed evolution of lead-binding proteins

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Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1 Contains the screening plasmid chassis cell construction of two-way screening system

[0044] Artificial Totally Synthetic Lead Binding Protein Gene pbrR and Promoter P pbr , whose nucleotide sequence is shown in SEQ ID No.1; ampicillin resistance gene amp, whose nucleotide sequence is shown in SEQ ID No.2; fructan sucrase gene sacB, whose nucleotide sequence is shown in Shown in SEQ ID No.3. The nucleotide sequences shown in SEQ ID No.1, SEQ ID No.2, and SEQ ID No.3 and the vector backbone gene fragments were connected by Gibson Assembly, and the positive clones were screened to obtain the screened plasmid. The plasmid map is as follows: figure 1 shown. The screening mechanism of the bidirectional screening system for directed evolution of lead-binding proteins such as figure 2 shown.

[0045] Use high-fidelity DNA polymerase to configure the reaction system, and amplify gene fragments by polymerase chain reaction: vector backbone gene fragments (prim...

Embodiment 2

[0048] The expression verification of the two-way screening system under the condition of metal lead ion of embodiment 2

[0049] The screening plasmid chassis cells containing the two-way screening system were inserted into LB liquid medium with kanamycin 50 μg / mL and cultured overnight at 37°C and 220 rpm, and then transferred to 50 mL LB liquid medium until the cells grew to OD 600 About 0.6.

[0050] (1) Expression verification of lead ion positive screening markers: the OD 600 About 0.6 bacteria solution was transferred to 50mL LB liquid medium supplemented with lead nitrate solution and ampicillin solution at a ratio of 1%, and the final concentration of lead ions was set to a concentration gradient of 0, 1, 5, 10, 20, and 50 μM , the final concentration of ampicillin solution was 100 μg / mL, and three parallels were set for each sample. Shake flask culture was carried out at 37°C and 220rpm, and the absorbance OD at a wavelength of 600nm was measured with a UV-visible ...

Embodiment 3

[0054] The expression optimization of the two-way screening system under the condition of metal lead-zinc ion of embodiment 3

[0055] The screening plasmid chassis cells containing the two-way screening system were inserted into kanamycin-resistant LB medium and cultured overnight at 37°C and 220rpm, and then transferred to 50mL LB liquid medium until the cells grew to OD 600 About 0.6.

[0056] (1) Expression optimization of positive screening markers at the same concentration of lead and zinc ions: the OD 600 About 0.6 of the bacterial solution was transferred to 50 mL of LB liquid medium supplemented with 100 μg / mL of ampicillin at a ratio of 1%, and lead nitrate solution and zinc nitrate solution were added to the final concentrations of 20 μM and 50 μM respectively. parallel. Shake flask culture was carried out at 37°C and 220rpm, and the absorbance OD at a wavelength of 600nm was measured with a UV-visible spectrophotometer every 3h 600 , draw the growth curve, such ...

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Abstract

The invention provides a method for constructing a bidirectional screening system for the directional evolution of lead-binding proteins. The bidirectional screening system is formed by using the forward screening marker ampicillin resistance gene amp and the reverse screening marker fructan sucrase gene sacB. Protein PbrR and specific promoter P pbr expression regulation. According to the results of expression verification and expression optimization of the two-way screening system, the screening conditions for the binding of lead ions and lead-binding proteins are 50 μM of lead ions and 100 μg / mL of ampicillin; 10%, cells with strong binding to zinc ions inhibited growth due to sucrose sensitivity. The optimization conditions are pressure screening conditions for the directed evolution of lead-binding proteins, which are used to reduce the interference of non-target metal ions, zinc ions, and realize the specific directed evolution of lead-binding proteins; provide a new screening for the specific optimization of this type of metal regulatory proteins tool.

Description

technical field [0001] The invention relates to a method for constructing a bidirectional screening system for directed evolution of lead-binding proteins, which provides help for improving the specific binding of metal regulatory proteins. Background technique [0002] The lead-binding protein is derived from the lead resistance operon pbr of the cupriavidus metallidurans CH34 plasmid pMOL30, which can simultaneously realize the uptake, transfer and enrichment of lead ions, forming the main line of defense of the strain against lead ions. The lead-binding protein PbrR manipulates P pbr The lead ion-dependent induced transcription of the structural gene pbrABCD downstream of the promoter plays a key role in the regulation of lead ions. After the lead-binding protein PbrR is expressed, it forms a homodimer and binds to the operon DNA. When lead ions are present, it changes the conformation of the protein-DNA complex, causing the DNA to unwind and present an open structure, t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/65C12N15/31
CPCC12N15/63C12N15/65C07K14/195
Inventor 贾晓强赵婷婷
Owner TIANJIN UNIV
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