This year, Jilin_China added 90 basic parts to the registry, including heat-inducible RNA-based thermosensors, heat-repressible RNA-based thermosensors, cold-inducible RNA-based thermosensors, cold-repressible RNA-based thermosensors and two different types of sfGFP. We have characterized and measured all of these parts, calculated their melting temperatures by using mathematical modeling, and successfully built a SynRT toolkit that allows users to select the appropriate RNA-based thermosensors in artificial biological systems.
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) 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. 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. 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. 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 (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. 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.
You can see the experiment results in the improvement page. Click Here!
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
Part Name Part Number Heat-inducible RNA-based thermosensor-1 BBa_K2541001 Heat-inducible RNA-based thermosensor-2 BBa_K2541002 Heat-inducible RNA-based thermosensor-3 BBa_K2541003 Heat-inducible RNA-based thermosensor-4 BBa_K2541004 Heat-inducible RNA-based thermosensor-5 BBa_K2541005 Heat-inducible RNA-based thermosensor-6 BBa_K2541006 Heat-inducible RNA-based thermosensor-7 BBa_K2541007 Heat-inducible RNA-based thermosensor-8 BBa_K2541008 Heat-inducible RNA-based thermosensor-9 BBa_K2541009 Heat-inducible RNA-based thermosensor-10 BBa_K2541010 Heat-inducible RNA-based thermosensor-11 BBa_K2541011 Heat-inducible RNA-based thermosensor-12 BBa_K2541012 Heat-inducible RNA-based thermosensor-13 BBa_K2541013 Heat-inducible RNA-based thermosensor-14 BBa_K2541014 Heat-inducible RNA-based thermosensor-15 BBa_K2541015 Heat-inducible RNA-based thermosensor-16 BBa_K2541016 Heat-inducible RNA-based thermosensor-17 BBa_K2541017 Heat-inducible RNA-based thermosensor-18 BBa_K2541018 Heat-inducible RNA-based thermosensor-19 BBa_K2541019 Heat-inducible RNA-based thermosensor-20 BBa_K2541020 Heat-inducible RNA-based thermosensor-21 BBa_K2541021 Heat-inducible RNA-based thermosensor-25 BBa_K2541025 Heat-inducible RNA-based thermosensor-26 BBa_K2541026 Heat-inducible RNA-based thermosensor-27 BBa_K2541027 Heat-inducible RNA-based thermosensor-28 BBa_K2541028 Heat-inducible RNA-based thermosensor-29 BBa_K2541029 Heat-inducible RNA-based thermosensor-30 BBa_K2541030 Heat-inducible RNA-based thermosensor-31 BBa_K2541031 Heat-inducible RNA-based thermosensor-32 BBa_K2541032 Heat-inducible RNA-based thermosensor-33 BBa_K2541033 Heat-inducible RNA-based thermosensor-34 BBa_K2541034 Heat-inducible RNA-based thermosensor-35 BBa_K2541035 Heat-inducible RNA-based thermosensor-36 BBa_K2541036 Heat-inducible RNA-based thermosensor-37 BBa_K2541037 Heat-inducible RNA-based thermosensor-38 BBa_K2541038 Heat-inducible RNA-based thermosensor-39 BBa_K2541039 Heat-inducible RNA-based thermosensor-40 BBa_K2541040 Heat-inducible RNA-based thermosensor-41 BBa_K2541041 Heat-inducible RNA-based thermosensor-42 BBa_K2541042 Heat-inducible RNA-based thermosensor-43 BBa_K2541043 Heat-inducible RNA-based thermosensor-44 BBa_K2541044 Heat-inducible RNA-based thermosensor-45 BBa_K2541045 Heat-inducible RNA-based thermosensor-46 BBa_K2541046 Heat-inducible RNA-based thermosensor-47 BBa_K2541047 Heat-inducible RNA-based thermosensor-48 BBa_K2541048 Heat-inducible RNA-based thermosensor-49 BBa_K2541049 Heat-inducible RNA-based thermosensor-50 BBa_K2541050 Heat-inducible RNA-based thermosensor-51 BBa_K2541051 Heat-repressible RNA-based thermosensor-1 BBa_K2541101 Heat-repressible RNA-based thermosensor-2 BBa_K2541102 Heat-repressible RNA-based thermosensor-3 BBa_K2541103 Heat-repressible RNA-based thermosensor-4 BBa_K2541104 Heat-repressible RNA-based thermosensor-5 BBa_K2541105 Heat-repressible RNA-based thermosensor-6 BBa_K2541106 Heat-repressible RNA-based thermosensor-8 BBa_K2541108 Heat-repressible RNA-based thermosensor-9 BBa_K2541109 Heat-repressible RNA-based thermosensor-10 BBa_K2541110 Heat-repressible RNA-based thermosensor-11 BBa_K2541111 Heat-repressible RNA-based thermosensor-12 BBa_K2541112 Heat-repressible RNA-based thermosensor-13 BBa_K2541113 Heat-repressible RNA-based thermosensor-14 BBa_K2541114 Heat-repressible RNA-based thermosensor-15 BBa_K2541115 Heat-repressible RNA-based thermosensor-16 BBa_K2541116 Heat-repressible RNA-based thermosensor-17 BBa_K2541117 Heat-repressible RNA-based thermosensor-18 BBa_K2541118 Heat-repressible RNA-based thermosensor-19 BBa_K2541119 Heat-repressible RNA-based thermosensor-20 BBa_K2541120 Heat-repressible RNA-based thermosensor-21 BBa_K2541121 Heat-repressible RNA-based thermosensor-22 BBa_K2541122 Heat-repressible RNA-based thermosensor-23 BBa_K2541123 Cold-inducible RNA-based thermosensor-1 BBa_K2541301 Cold-inducible RNA-based thermosensor-2 BBa_K2541302 Cold-inducible RNA-based thermosensor-3 BBa_K2541303 Cold-inducible RNA-based thermosensor-4 BBa_K2541304 Cold-inducible RNA-based thermosensor-5 BBa_K2541305 Cold-inducible RNA-based thermosensor-6 BBa_K2541306 Cold-inducible RNA-based thermosensor-7 BBa_K2541307 Cold-inducible RNA-based thermosensor-8 BBa_K2541308 Cold-repressible RNA-based thermosensor-1 BBa_K2541201 Cold-repressible RNA-based thermosensor-2 BBa_K2541202 Cold-repressible RNA-based thermosensor-3 BBa_K2541203 Cold-repressible RNA-based thermosensor-4 BBa_K2541204 Cold-repressible RNA-based thermosensor-5 BBa_K2541205 Cold-repressible RNA-based thermosensor-6 BBa_K2541206 Cold-repressible RNA-based thermosensor-7 BBa_K2541207 Cold-repressible RNA-based thermosensor-8 BBa_K2541208 Cold-repressible RNA-based thermosensor-9 BBa_K2541209 Cold-repressible RNA-based thermosensor-10 BBa_K2541210
- Kortmann J, Narberhaus F. Bacterial RNA thermometers: molecular zippers and switches.[J]. Nature Reviews Microbiology, 2012, 10(4):255-65.
- Pertzev A V, Nicholson A W. Characterization of RNA sequence determinants and antideterminants of processing reactivity for a minimal substrate of Escherichia coli ribonuclease III[J]. Nucleic Acids Research, 2006, 34(13):3708-3721.
- Giuliodori A M, Di P F, Marzi S, et al. The cspA mRNA is a thermosensor that modulates translation of the cold-shock protein CspA.[J]. Molecular Cell, 2010, 37(1):21-33.
- Breaker R R. RNA Switches Out in the Cold[J]. Molecular Cell, 2010, 37(1):1-2.
- Overkamp W, Beilharz K, Detert O W R, et al. Benchmarking various green fluorescent protein variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for live cell imaging.[J]. Applied & Environmental Microbiology, 2013, 79(20):6481-6490.