DNA composite hydrogel and preparation method thereof

Abstract

The invention relates to the technical field of biology, in particular to DNA composite hydrogel and a preparation method thereof. The preparation method comprises the following steps of uniformly mixing 0.5-4% by mass of clay, 0.2-30% by mass of a functional group-containing high-molecular compound and 10-400 ng / mu L of a DNA template at a relatively low temperature, and polymerizing at a relatively high temperature to form the DNA composite hydrogel. The DNA composite hydrogel provided by the invention can protect the DNA template, and can also improve the local effective concentration of the DNA template in acellular protein expression, thereby improving the protein yield. Meanwhile, the hydrogel can be repeatedly used for multiple times, so that the reaction cost is further reduced.

Description

technical field [0001] The application relates to the field of biotechnology, in particular to a DNA composite hydrogel for cell-free protein expression and a preparation method thereof. Background technique [0002] Most of the existing protein preparation methods use cells as hosts to synthesize in living cells, but they have many disadvantages: cumbersome operations, long cycle times, and extremely limited types of proteins to be synthesized. Cell-free protein synthesis (Cell-free Protein Synthesis, CFPS) system is an in vitro gene expression system that uses exogenous DNA or mRNA as a template, artificially adds the required raw materials and energy substances, and synthesizes proteins under the condition of cell extracts, which can break through the limitations of cells. Express various proteins conveniently and quickly. In detail, CFPS uses DNA as a template to transcribe corresponding mRNA under the action of RNA polymerase and transcription factors; using mRNA as a ...

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Problems solved by technology

[0002] Most of the existing protein preparation methods use cells as hosts to synthesize in living cells, but they have many disadvantages: cumbersome operations, long cycle times, and extremely limited types of synthesized proteins, etc.

(2) Using a strain that has knocked out the gene encoding endonuclease E (RNase E) to prepare cell extracts delayed the degradation of mRNA molecules, and the protein levels expressed from LETs amplified by PCR became comparable to conventional plasmid-based methods. The response is comparable, but the problem is that the strain grows slowly and takes time

(3) By designing special primers, DNA linear fragments are formed into circular DNA molecules similar to plasmids, which improves the resistance of target gene fragments to exonucleases, but is expensive

(4) Luo's research group applied the DNA hydrogel system to CFPS, mainly linking linear template DNA molecules with X-shaped DNA (X-DNA) to generate DNA hydrogels for combined cell-free transcription and translation systems. Glue; in a wheat germ cell-free system, this method resulted in a 300-fold increase in protein yield compared to soluble DNA templates, however X-DNA synthesis is expensive

Although the clay hydrogel is cheap, it has thixotropy and is easily broken during the shaking process, which limits the reuse of the hydrogel template.

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