Difference between revisions of "Team:Jilin China/Basic Part"

RNA-based thermosensors

Heat-inducible RNA-based thermosensors

Heat-inducible RNA-based thermosensors are RNA genetic control systems that sense temperature changes. At low temperatures, the mRNA adopts a stem-loop conformation that masks the Shine–Dalgarno (SD) sequence within the 5’-untranslated region (5’-UTR)^[1] and, in this way, prevents ribosome binding and translation. At elevated temperatures, the RNA secondary structure melts locally, thereby giving the ribosomes access to the SD sequence to initiate translation. Whereas natural RNA-based thermosensors have a relatively complicated secondary structure with multiple stems, hairpin loops and bulges which impeds application process. Our team designed synthetic heat-inducible RNA-based thermosensors that are considerably simpler than naturally occurring thermosensors and can be exploited as convenient on/off switches of gene expression. Since they performed very well, we chose BBa_K2541029 as our favorite basic part, which was the most extraordinary one.

Heat-repressible RNA-based thermosensors

We designed short, heat-repressible RNA-based thermosensors. These thermosensor sequences contain a single-strand RNase E cleavage (RC) site. RNase E is an enzyme native to Escherichia coli and many other organisms^[2]. Each heat-repressible RNA-based thermosensor sequence was inserted downstream of the transcription start site and upstream of the SD sequence. At high temperatures, the RC is exposed, mRNA was cleaved by RNase E, and expression is ‘OFF’. At low temperatures, the RC binds to the anti-RNase E cleavage site (ARC) and forms a stem-loop. This structure sequesters the RC, and expression is ‘ON’.

Cold-inducible RNA-based thermosensors

There are multiple families of cold-inducible proteins in prokaryotes, the most widely studied of which are the Csp family of cold shock proteins in E.coli. CspA represents CspA family, which has been quite extensively studied for the mechanism of its cold response. There is a temperature-sensing region in the 5'UTR of CspA mRNA, which can regulate the accessibility of the translation initiation region by altering the advanced structure of RNA, thereby regulating the initiation of translation[3]. At low temperatures (<20℃), 5’UTR of CspA mRNA can form an advanced structure called pseudoknot, which is more efficiently translated because the conformation exposes the SD sequence, it is beneficial to recruit ribosomes and somewhat less susceptible to degradation. At normal temperatures, due to thermodynamic instability, pseudoknot unfolds. 5’UTR forms a secondary structure masking SD sequence to block translation initiation region, which impedes translation. We designed a series of cold-inducible RNA-based thermosensors with different melting temperatures, intensity and sensitivity based on the pseudoknot structure.

Cold-repressible RNA-based thermosensors

This year, we designed short, cold-repressible RNA-based thermosensors, which will form a stem-loop upstream of the SD sequence. These thermosensor sequences contain a double-strand RNA cleavage site for RNase III, an enzyme native to Escherichia coli and many other organisms^[4]. At low temperatures, the mRNA stem-loop is stable to expose the RNase III cleavage site and the transcript will be degraded. At elevated temperatures, the stem-loop will unfold and translation will occur unhindered.

sfGFP_optimism (BBa_K2541400)

sfGFP (superfolder GFP), whose emission and excitation wavelength are similar to GFP, contains a higher fluorescence intensity and folding speed than GFP. Thus, we applied sfGFP as the reporter protein in our measurement device^[5]. However, the existing sfGFP (BBa_I746916) in the registry contains a BbsI restriction site, and BbsI restriction endonuclease is an economical and efficient enzyme used in Golden Gate assembly, so the sfGFP (BBa_I746916) cannot be used for Golden Gate assembly. In view of that, we designed a site-directed mutation of sfGFP (BBa_K2541401) by creating a double-base mutation to the BbsI recognition site without changing the amino acid sequence. sfGFP BBa_K2541401 won’t be digested during the assembly, so we called it sfGFP(BbsI free).

We also performed a codon optimization sfGFP and named it sfGFP_optimism. Then we designed a composite part, which consists of Anderson Promoter J23104, RBS B0034, sfGFP_optimism and double terminator B0010 and B0012. We did experiments to compare it with sfGFP and sfGFP(BbsI free). Our results showed that the sfGFP_optimism has a higher fluorescence intensity than others, so we finally chose sfGFP_optimism as our reporter protein.

Since sfGFP has more advantages than GFP, and Golden Gate assembly will be used by more researchers as an efficient and scarless assembly method in the future, so we decided to add sfGFP_optimism to the parts registry.

Basic parts content