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Method for reinforcing in vitro genetic transcription and translation efficiency

A gene transcription and enhancer technology, which is applied in the introduction of foreign genetic material using vectors, recombinant DNA technology, etc., can solve the problems of low in vitro gene transcription and translation efficiency, and achieve enhanced efficiency and protein yield, shorten reaction time, and improve efficiency. Effect

Inactive Publication Date: 2009-07-08
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the deficiencies in the prior art, the present invention provides a method for enhancing the efficiency of in vitro gene transcription and translation, and directly adds the prepared gold nanoparticles (5nm to 40nm) of different sizes into in vitro gene transcription and translation reagents to solve the problem of in vitro gene transcription. The problem of low translation efficiency

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0018] 2. Preparation of colloidal gold

[0019] Take 50mL of HAuCl with a mass fraction of 0.01% 4 Solution (dissolve 0.01g HAuCl in 100mL water 4 ), heated to reflux until boiled, quickly added different volumes of 350 μL and 330 μL trisodium citrate solutions (mass fraction: 1%) under vigorous stirring, and continued to react for 15 minutes after the color changed, then stopped the reaction, cooled at room temperature, and obtained 30nm, 40nm gold nanoparticles.

[0020] 3. Preparation of smaller gold nanoparticles by changing HAuCl 4 solution and trisodium citrate solution, the HAuCl 4 The solution is added to the boiling trisodium citrate solution.

[0021] Example: Dissolve 68mg of trisodium citrate in 105mL of water, heat to reflux until boiling, and quickly add 1mL of HAuCl under vigorous stirring 4 Solution (containing 9.5mg HAuCl 4 ) after the color changes, continue to react for 15 min, 10 min, and 5 min, then terminate the reaction, cool at room temperature, ...

Embodiment 1

[0023] In Example 1, 5nm gold particles and E coli RTS100 kit were selected to enhance the efficiency of in vitro gene transcription and translation and increase protein production. Take the following steps:

[0024] 1. Preparation of in vitro gene transcription and translation reaction reagents containing gold nanoparticles:

[0025] Take 7 500μl PCR reaction tubes, add 12μl E.Coli lysate, 10μl reaction solution, 12μl 20 kinds of amino acid mixture, 1μl methionine, 5μl reconstitution buffer, 9μl brcaal gene template in order, and then add 0.12nM , 0.24nM, 0.48nM, 0.72nM, 0.96nM, 1.2nM 5nm gold particles into different reaction tubes, then, add RNase-free water, another tube without gold nanoparticles, as a control, until the total volume of 50μl.

[0026] 2. Reagent reaction tubes containing gold nanoparticles and target genes are reacted on the instrument:

[0027] The reaction tube containing the gold-containing particles and the RTS reaction solution prepared in step 1 w...

Embodiment 2

[0030] In Example 2, 20nm gold particles and E coli RTS100 kit were selected to enhance the efficiency of gene transcription and translation in vitro and increase protein production. Take the following steps:

[0031] 1. Preparation of in vitro gene transcription and translation reaction reagents containing gold nanoparticles:

[0032] Take 7 500μl PCR reaction tubes, add 12μl E.Coli lysate, 10μl reaction solution, 12μl 20 kinds of amino acid mixture, 1μl methionine, 5μl reconstitution buffer, 9μl brcaal gene template in order, and then add 0.12nM , 0.24nM, 0.48nM, 0.72nM, 0.96nM, 1.2nM 20nm gold particles into different reaction tubes, then, add RNase-free water, another tube without gold nanoparticles, as a control, until the total volume of 50μl.

[0033] 2. Reagent reaction tubes containing gold nanoparticles and target genes are reacted on the instrument:

[0034] The reaction tube containing the gold-containing particles and the RTS reaction solution prepared in step 1...

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Abstract

The present invention relates to a method for enhancing in vitro gene transcription and translation efficiency enhancement in the field of biological engineering technique, specifically including: randomly mixing the gold nanometer particles between 5nm to 40nm or them, adding in the in vitro gene transcription and translation reagent box, wherein, the final concentration of the gold nanometer particle is controlled between1.2nM to 0.12nM, using conventional reaction conditions to obtain protein product having a higher reaction than the nanometer particles without gold, for enhancing the efficiency of in vitro gene transcription and translation. The inventive has simple method, low cost, and application in large-scale protein preparation, regulation of gene transcription and translation, toxic protein preparation, structure and function investigation. Conventional biochemical labs all has the experiment conditions, thereby facilitating promotion.

Description

technical field [0001] The invention relates to a high-efficiency protein preparation method in the technical field of bioengineering, in particular to a method for enhancing gene transcription and translation efficiency in vitro. Background technique [0002] Conventional protein preparation technology is to clone gene fragments into protein expression vectors, and then transfer them into Escherichia coli DH5α or insects or yeasts for induced expression, and then, cleavage and isolate and purify the expressed proteins. This method is relatively complicated, and the efficiency of obtaining protein products is relatively low. More importantly, sometimes the products are inactive. Subsequently, Roche and others launched an in vitro rapid translation system kit (Rapid Translation System, RTS), which can prepare milligram-level proteins within 5 hours. This system employs a coordinated transcription-translation reaction for in vitro protein synthesis, in which T7 RNA polymerase...

Claims

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Application Information

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IPC IPC(8): C12N15/67
Inventor 崔大祥李清高峰
Owner SHANGHAI JIAO TONG UNIV