Synthetic Promoters Construction
With the development of synthetic biology, scientists have set a goal to construct de novo biobricks, parts and systems, while realize the strandardization of biological system. De novo genetic biobricks are of great value in metabolic engineering and systems biology, especially. Promoters represent critical elements that can participate in gene expression and regulation, which play an important role in artificial systems. However, promoters from different sources have sophisticated regulation system, low universality, unpredictable expression intensity and lack of standardization, their applications in synthetic biology have been impeded. Fortunately, de novo promoters construction and identification have provided a brand new method to address this issue.
A prokaryotic promoter is made up of -35 region, -10 region, spacers, UP element as conservative regions and other non-conserved regions. According to current researches, a promoter is also considered as core promoter with its downstream sequence including 5’Untranslated Region (5’UTR). Strictly, promoters and ribosomes binding sites (RBS) are regulatory elements of transcription and translation respectively. Because of the short sequence of RBS, we connect synthetic RBS library to the library of synthetic core promoters, then a novel library of promoters can be constructed.
Temperature-induced Promoter Library Construction
Compared to widely used chemically induced promoters, physically induced promoters don’t require special inducer, reducing cost while avoiding super-sensitivity of inducer concentration. Besides, physically induced promoters have special characteristics and a vast application prospect, and among all kinds of physical factors, temperature has become our main concern this year with its vast prospect in fermentation engineering, drug targeting and other fields. As a result, our project, with a view to construct and characterize synthetic regulated Temperature-Sensitive promoter libraries for 2018 iGEM competition.
According to current researches, there are three different types of biomolecules responsive to temperature: DNA, RNA and proteins. Actually, RNA is playing a dominant role in temperature sensing especially in bacteria. 5’UTR of mRNA forms secondary structure, in order to block or expose SD （Shine-Dalgarno sequence，binding to ribosomes in prokaryote cells）sequence. Compared to DNA and proteins, it is easier to reconstruct temperature-sensing RNA.
Our primary objective is to construct a temperature-sensitive promoter library supplying the iGEM registry with temperature-sensitive promoter parts collection. However, natural temperature-sensitive promoters, which are in a small number lacking in standardization, are regulated by many trans-acting factors, we try to design and construct de novo synthetic temperature-sensitive promoters with current researches.
What are we doing?
We construct our promoter library with the free combination of core promoter and 5＇UTR, then the expression efficiency is easily to be changed with the replacement of different core promoters and sequence changes of 5’UTR, realizing the bi-level regulation of both transcription and translation. Four synthetic standard regulated libraries have been constructed:
- (1) Heat-induced RNA thermosensors library, directional reconstructions to secondary structures of RNA thermometers;
- (2) Heat-repressible RNA thermosensors library, directional reconstructions to anti-RNaseE cleavage site;
- (3) Cold-induced RNA thermosensors library, directional reconstructions to mRNA pseudoknot of CspA;
- (4) Cold-repressible RNA thermosensors library, directional reconstructions to RNase III binding site.