First of all, the accuracy of decoding mRNA can be precisely regulated by manipulating the tRNA population added to the CFPS. Simplified codon protein synthesis offers several profound advantages for the bioengineering and study of protein synthesis ( Figure 1). To establish a simplified codon set, two conditions are required in the protein synthesis reaction system: 1) the expression template is simplified to only one codon corresponding to one tRNA 2) the orthogonality of the tRNA/aaRS/AA system needs to be maintained. To reduce the redundancy of the genetic codon, it is necessary to expand the coding scope by engineering these orthogonal triplets inside a cell or in vitro. It is well known that different codons with their cognate tRNAs and aminoacyl-tRNA synthetases (aaRS) act orthogonally during protein translation. During protein synthesis, the aminoacylated tRNAs can bring the specific amino acid to a cognate codon of the mRNA inside the ribosome, which then elongates the polypeptide ( Su et al., 2020). As carriers of amino acids, tRNAs play a vital role in converting the genetic information from messenger RNA (mRNA) into the polypeptide chain (proteins). This degeneracy of the genetic codon provides favorable conditions for us to rearrange the orthogonal sense codons ( Li, 2021).
Therefore, there is redundancy in the genetic codon, so that some amino acids are encoded by several so-called synonymous codons. In general, there are 64 different combinations of the four nucleotides that encode a pool of 20 amino acids and translation stop signals in the genetic codon. It is hoped that the tRNA complement protein synthesis system can facilitate the construction of minimal cells and expand the biomedical application scope of synthetic biology.Ī codon constitutes three consecutive nucleotide bases that are recognized by a specific tRNA to integrate a single amino acid into a growing peptide chain inside the ribosome, or a stop signal that terminates protein synthesis. Furthermore, we point out the trend toward a minimized genetic codon for reducing codon redundancy by manipulating tRNAs in the different proteins. Strategies for removing tRNAs from cell lysates and synthesizing tRNAs in vivo/ vitro are summarized and discussed in detail. These designed polypeptide coding sequences reduce the genetic codon and contain only a single tRNA corresponding to a single amino acid in this presented system. Here, we provide an overview of the tRNA complement protein synthesis system construction in the tRNA-depleted Protein synthesis Using purified Recombinant Elements (PURE) system or S30 extract. Manipulating transfer RNAs (tRNAs) for emancipating sense codons to simplify genetic codons in a cell-free protein synthesis (CFPS) system can offer more flexibility and controllability.
2Frontiers Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin, China.
1School of Chemical Engineering and Technology, Tianjin University, Tianjin, China.Jiaojiao Li 1,2, Mengtong Tang 1,2 and Hao Qi 1,2*
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