Team:GIFU TOKAI/Background
Background
Basic information of our project
Cell-free system
Cell-free transcription-translation system
Cell-free protein synthesis is a valuable technique for synthetic biology. The demand for alternative and highly effective in vitro expression systems is increasing as more strategies are developed. Cell-free systems include almost all protein synthesis factors. Proteins can be provided just by attending DNA/RNA to protein synthesis factors. For the last decade, cell-free system’s efficiency and utility value have been improved.
An all Escherichia coli cell-free system
An all Escherichia coli was taken advantage of produce T7 phages, including DNA replication and packaging, explaining the potential of cell-free transcription-translation to construct complicated systems from scratch with genome-sized DNA programs. Ribosomes were compounded using hybrid T7-based and other transcription factor-based in vitro transcription-translation. Such achievements, performed with kits able to produce 1mg/ml of protein, promote the development of more mighty in vitro transcription-translation. According to the latest information, using maltose as a metabolite, E-coli system derivers protein efficiently through a single bacterial promoter. An all E.coli cell-free system, which uses the endogenous transcription and molecular translation system, is optimized to synthesize up to 2.3 mg/ml of a reporter protein in batch mode reactions. myTXTL from Arbor Biosciences belong to this type.
PURE system
Using all E.coli extracts logically encounters two problems: a rapid depletion of energy charge, independent of peptide bond formation, and decline of protein products or template nucleic acids by proteases or nucleases. On the other hand, the approach attempting to reconstitute protein synthesis from purified components of the translation overcomes some matters of the system using all E.coli extracts. The PURE system contains all necessary translation factors. The PURE system includes 32 components: IF1, IF2, IF3, EF-G, EF-Tu, IF-Ts, RF1, RF3, RRF, 20 aminoacyl-tRNA synthetases (ARSs), methionyl-tRNA formyltransferase (MTF), T7 RNA polymerase, and ribosomes. Additionally, the system contains 46 tRNAs, NTPs, creatine phosphate, 10-formyl-5,6,7,8-tetrahydrofolic acid, 20 amino acids, creatine kinase, myokinase, nucleoside-diphosphate kinase, and pyrophosphatase. They are purified with particularly high activity and enable efficient protein production. The PURE system was able to produce protein at a rate of about 160 μg/ml/h in a batch mode without the necessity for any supplementary apparatus. This system is also expedient in terms of product purification. The PURE system can insert an amino acid coinciding with a particular termination codon, that it can be applied to synthesize proteins containing unnatural amino acids. Ultimately PURE system for reconstituting translation from purified translation factors is an efficient way of producing active proteins from template nucleic acids in considerable amounts. PUREfrex from GeneFrontier belongs to this type.
Summary
Each group of the cell-free system has each characteristic. Based on what experimenters want to do, the systems should be selected.
Reference
1. Filippo Caschera, Vincent Noireaux. Synthesis of 2.3mg/ml of protein with an all Escherichia coli cell-free transcription-translation system. Biochimie.99,162-168(2014)
2. Yoshio Shimizu, Akio Inoue, Yukihide Tomari, Tsutomu Suzuki, Takahashi Yokogawa, Kazuya Nishikawa, and Takuya Ueda. Cell-free translation reconstituted with purified components. Nature biotechnology. 19 (2001)
For further information about this theme, please visit the websites below.
Arbor Biosciences GeneFrontierTranslation coupling
Translation coupling of operon in prokaryotic cells
It is commonly known that prokaryote does not have the process of nuclear-cytoplasmic interaction. Therefore, DNA synthesis, RNA synthesis and translation into protein happen simultaneously. Generally, bacterial genes are transcribed to form polycistronic mRNAs bearing reading frames which its translational efficiencies are not determined independently. The mechanism of translation coupling causes this phenomenon. As one type of the results of the translation coupling reaction, it is clear that genes that locate upstream are translated efficiently than downstream genes. Its mRNA has multiple translational initiation sites, and this enables bacterial genes to translate multiple types of proteins from one mRNA. Gene cluster of operon like E-coli’s Tryptophan operon is one of the examples.
Tryptophan, which is a kind of amino acid, is synthesized by several steps, and the enzymes that catalyze those steps form an operon. Cells suppress Tryptophan operon at ordinary times, but when the supply of the Tryptophan exceeds the demand, Tryptophan binds with the repressor and the repressor protein-tryptophan complex will be able to bind with the operator site which inhibits RNA polymerase to bind with the promoter site. In this operon, translation of the two genes (trpBA) is coupled and according to the paper from SERAP AKSOY, 83% of trpA translation is dependent on atpB mRNA which means the gene located upstream is translated efficiently than the downstream gene. The difference of translation efficiency indicates that translational coupling occurs in trp operon of E.coli.
When translation step is completed in E.coli, the small subunit of ribosome identifies the start codon, and the large subunit binds with the small subunit. At the end of the translation, ribosome identifies the stop codon, and it dissociates from mRNA. However, by putting a specific region between operons downstream the stop codon, it is possible to make ribosome not dissociated. Technically, ribosomes can keep binding to mRNA after facing stop codon in some cases.
In the previous reports, translation coupling is expected to be possible when there is a stem-loop structure or the length between the start codon and the stop codon is less than approximately four bp: when the distance between the start codon and the stop codon is physically close. From this point, one paper from Inokuchi demonstrated that it is possible to synthesize two kinds of protein; RNA phase GA coat and lysis protein from one RNA, though it is not a gene cluster. When the upstream sequence is eliminated, the expression of the downstream lysis gene was abolished entirely. Moreover, when the Shine-Dalgarno sequence was introduced in this construct, the expression of the lysis gene was restored without the coat gene.
Following this hypothesis and theory, iGEM GIFU TOKAI conducted iVEPOP, in vitro eternal expression of protein, by utilizing translation coupling inside the circular RNA.
Figure.1 Translation coupling