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Construction method of temperature-controlled cell-free reaction system, plasmid used by method and application of plasmid

A reaction system, cell-free technology, applied in the direction of using vectors to introduce foreign genetic material, other methods of inserting foreign genetic material, medical preparations of non-active ingredients, etc., can solve the problem of effective control of protein expression, cell metabolism intensity and metabolism Flow changes, adverse effects of target pathways, etc., to achieve good temperature control and low cost effects

Pending Publication Date: 2022-06-28
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the aforementioned intracellular regulatory mechanisms in response to temperature changes face some inherent difficulties.
Temperature is an important factor affecting enzyme activity and complex cellular metabolism, and changes in temperature may lead to changes in cellular metabolic intensity and metabolic flux, thereby adversely affecting target pathways
Furthermore, interactions between different synthetic pathways in vivo may limit the effective control of protein expression

Method used

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  • Construction method of temperature-controlled cell-free reaction system, plasmid used by method and application of plasmid
  • Construction method of temperature-controlled cell-free reaction system, plasmid used by method and application of plasmid
  • Construction method of temperature-controlled cell-free reaction system, plasmid used by method and application of plasmid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Example 1: Plasmid construction

[0122] The bacteriophage lambda repressor cI is a well-known temperature-sensitive variant that acts on the tandem pR-pL operator-promoter and can efficiently regulate genes cloned downstream of the promoter. When the temperature is below 37°C (usually 28-32°C), it represses gene expression, while when the host RNA polymerase raises the temperature above 37°C, the mutated repressor is inactivated and transcribed, the regulation mechanisms such as figure 2 shown.

[0123] All plasmids used in the present invention were constructed according to standard molecular biology techniques.

[0124] In the present invention, the pSB3K3 plasmid (purchased from Addgene (Addgene#78636)37) is used as the backbone, and the green fluorescent protein GFP is selected as the reporter protein (synthesized by GENEWIZ company). The fluorescent reporter gene was cloned by Gibson assembly under the control of the pL / pR promoter, and the expression suppress...

Embodiment 2

[0132] Example 2: Expression of plasmids in cell systems

[0133] The pcI plasmid and pΔcI plasmid were transformed into E. coli BL21 Star (DE3) and cultured at 30°C and 37°C for 12 hours, respectively. The OD600 and fluorescence intensity of the bacterial solution were then determined. The result is as Figure 4 As shown, the GFP level of the pcI plasmid at 37°C was higher than that at 30°C, confirming that the constructed pcI plasmid could activate the expression of the target protein at the expected high temperature in the cells.

Embodiment 3

[0134] Example 3: Expression of plasmids in cell-free systems

[0135] The protein synthesis control ability of tcCFPS was tested by adding the pcI plasmid directly to the cell-free system and initiating the reaction at different temperatures.

[0136] - Preparation of Escherichia coli BL21 Star (DE3) cell-free extract (abbreviated as BS)

[0137] For overnight culture with E. coli, 200 mL of 2×YT-P medium was inoculated into a 1-L flask at a dilution ratio of 1:20. The culture was added to a 4 l fermenter and incubated at 37°C, 500 rpm for 3.5 hours. Cell beads were washed twice with 100 mL of ice-cold S30A buffer (14 mM mg-glutamic acid, 60 mM k-glutamic acid, 50 mM Tris, pH 7.7) and then centrifuged at 8000 xg for 15 minutes at 4°C. Then, the cell microspheres were resuspended in 30 ml of frozen S30A buffer, transferred to a pre-weighed 50 ml Falcon conical tube, and centrifuged at 10,000 × g for 10 minutes at 4°C. Finally, the tubes were reweighed and snap-frozen in nit...

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Abstract

The present invention relates to a plasmid comprising: a gene encoding a temperature sensitive element; the control unit is positioned at the downstream of the gene for coding the temperature sensitive element and can be regulated and controlled by the temperature sensitive element; and the exogenous gene is positioned at the downstream of the manipulation unit and is used for expressing the target protein. The plasmid can regulate and control expression of target protein through the control unit regulated and controlled by the temperature sensitive element, when the temperature rises to a given temperature, the temperature sensitive element is inactivated, so that the control unit starts expression of the target protein, and when the temperature is lower than the given temperature, the control unit starts expression of the target protein. The temperature-sensitive element inhibits the manipulation unit from initiating expression of the target protein. The invention further provides a construction method of a temperature-controlled cell-free synthesis system through the plasmid, and expression of target protein in the cell-free system is regulated and controlled through temperature.

Description

technical field [0001] The invention belongs to the field of biological reaction systems, and in particular relates to a construction method of a temperature-controlled cell-free reaction system, plasmids used in the method and applications. Background technique [0002] Bioprocess control is an important issue in the fields of biomanufacturing and genetic engineering. The spatiotemporal activation and regulation of protein synthesis and site-specific biological functions are of great significance in the fields of biotechnology, medicine, and industrial production. A commonly used control method is to use the chemical effects of molecules to regulate gene expression. However, this method also suffers from some disadvantages, including unstable chemical effects\low spatial precision and other undesirable side effects that chemical inducers may bring. Using physical signals such as light and heat to control biological processes is an ideal approach. Compared with the poor p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/87C12P21/02A61K9/127A61K47/46A61K47/42A61K47/28A61K47/24A61K38/16
CPCC12N15/63C12N15/87C12P21/02A61K9/127A61K47/46A61K47/42A61K47/28A61K47/24A61K38/16
Inventor 卢元杨俊祝汪琛
Owner TSINGHUA UNIV
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