Team:Jilin China/Basic Part


Basic Part

  • Abstract

    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.

  • 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.

    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 NamePart Number
    Heat-inducible RNA-based thermosensor-1BBa_K2541001
    Heat-inducible RNA-based thermosensor-2BBa_K2541002
    Heat-inducible RNA-based thermosensor-3BBa_K2541003
    Heat-inducible RNA-based thermosensor-4BBa_K2541004
    Heat-inducible RNA-based thermosensor-5BBa_K2541005
    Heat-inducible RNA-based thermosensor-6BBa_K2541006
    Heat-inducible RNA-based thermosensor-7BBa_K2541007
    Heat-inducible RNA-based thermosensor-8BBa_K2541008
    Heat-inducible RNA-based thermosensor-9BBa_K2541009
    Heat-inducible RNA-based thermosensor-10BBa_K2541010
    Heat-inducible RNA-based thermosensor-11BBa_K2541011
    Heat-inducible RNA-based thermosensor-12BBa_K2541012
    Heat-inducible RNA-based thermosensor-13BBa_K2541013
    Heat-inducible RNA-based thermosensor-14BBa_K2541014
    Heat-inducible RNA-based thermosensor-15BBa_K2541015
    Heat-inducible RNA-based thermosensor-16BBa_K2541016
    Heat-inducible RNA-based thermosensor-17BBa_K2541017
    Heat-inducible RNA-based thermosensor-18BBa_K2541018
    Heat-inducible RNA-based thermosensor-19BBa_K2541019
    Heat-inducible RNA-based thermosensor-20BBa_K2541020
    Heat-inducible RNA-based thermosensor-21BBa_K2541021
    Heat-inducible RNA-based thermosensor-25BBa_K2541025
    Heat-inducible RNA-based thermosensor-26BBa_K2541026
    Heat-inducible RNA-based thermosensor-27BBa_K2541027
    Heat-inducible RNA-based thermosensor-28BBa_K2541028
    Heat-inducible RNA-based thermosensor-29BBa_K2541029
    Heat-inducible RNA-based thermosensor-30BBa_K2541030
    Heat-inducible RNA-based thermosensor-31BBa_K2541031
    Heat-inducible RNA-based thermosensor-32BBa_K2541032
    Heat-inducible RNA-based thermosensor-33BBa_K2541033
    Heat-inducible RNA-based thermosensor-34BBa_K2541034
    Heat-inducible RNA-based thermosensor-35BBa_K2541035
    Heat-inducible RNA-based thermosensor-36BBa_K2541036
    Heat-inducible RNA-based thermosensor-37BBa_K2541037
    Heat-inducible RNA-based thermosensor-38BBa_K2541038
    Heat-inducible RNA-based thermosensor-39BBa_K2541039
    Heat-inducible RNA-based thermosensor-40BBa_K2541040
    Heat-inducible RNA-based thermosensor-41BBa_K2541041
    Heat-inducible RNA-based thermosensor-42BBa_K2541042
    Heat-inducible RNA-based thermosensor-43BBa_K2541043
    Heat-inducible RNA-based thermosensor-44BBa_K2541044
    Heat-inducible RNA-based thermosensor-45BBa_K2541045
    Heat-inducible RNA-based thermosensor-46BBa_K2541046
    Heat-inducible RNA-based thermosensor-47BBa_K2541047
    Heat-inducible RNA-based thermosensor-48BBa_K2541048
    Heat-inducible RNA-based thermosensor-49BBa_K2541049
    Heat-inducible RNA-based thermosensor-50BBa_K2541050
    Heat-inducible RNA-based thermosensor-51BBa_K2541051
    Heat-repressible RNA-based thermosensor-1BBa_K2541101
    Heat-repressible RNA-based thermosensor-2BBa_K2541102
    Heat-repressible RNA-based thermosensor-3BBa_K2541103
    Heat-repressible RNA-based thermosensor-4BBa_K2541104
    Heat-repressible RNA-based thermosensor-5BBa_K2541105
    Heat-repressible RNA-based thermosensor-6BBa_K2541106
    Heat-repressible RNA-based thermosensor-8BBa_K2541108
    Heat-repressible RNA-based thermosensor-9BBa_K2541109
    Heat-repressible RNA-based thermosensor-10BBa_K2541110
    Heat-repressible RNA-based thermosensor-11BBa_K2541111
    Heat-repressible RNA-based thermosensor-12BBa_K2541112
    Heat-repressible RNA-based thermosensor-13BBa_K2541113
    Heat-repressible RNA-based thermosensor-14BBa_K2541114
    Heat-repressible RNA-based thermosensor-15BBa_K2541115
    Heat-repressible RNA-based thermosensor-16BBa_K2541116
    Heat-repressible RNA-based thermosensor-17BBa_K2541117
    Heat-repressible RNA-based thermosensor-18BBa_K2541118
    Heat-repressible RNA-based thermosensor-19BBa_K2541119
    Heat-repressible RNA-based thermosensor-20BBa_K2541120
    Heat-repressible RNA-based thermosensor-21BBa_K2541121
    Heat-repressible RNA-based thermosensor-22BBa_K2541122
    Heat-repressible RNA-based thermosensor-23BBa_K2541123
    Cold-inducible RNA-based thermosensor-1BBa_K2541301
    Cold-inducible RNA-based thermosensor-2BBa_K2541302
    Cold-inducible RNA-based thermosensor-3BBa_K2541303
    Cold-inducible RNA-based thermosensor-4BBa_K2541304
    Cold-inducible RNA-based thermosensor-5BBa_K2541305
    Cold-inducible RNA-based thermosensor-6BBa_K2541306
    Cold-inducible RNA-based thermosensor-7BBa_K2541307
    Cold-inducible RNA-based thermosensor-8BBa_K2541308
    Cold-repressible RNA-based thermosensor-1BBa_K2541201
    Cold-repressible RNA-based thermosensor-2BBa_K2541202
    Cold-repressible RNA-based thermosensor-3BBa_K2541203
    Cold-repressible RNA-based thermosensor-4BBa_K2541204
    Cold-repressible RNA-based thermosensor-5BBa_K2541205
    Cold-repressible RNA-based thermosensor-6BBa_K2541206
    Cold-repressible RNA-based thermosensor-7BBa_K2541207
    Cold-repressible RNA-based thermosensor-8BBa_K2541208
    Cold-repressible RNA-based thermosensor-9BBa_K2541209
    Cold-repressible RNA-based thermosensor-10BBa_K2541210
  • References

    • [1]Kortmann J, Narberhaus F. Bacterial RNA thermometers: molecular zippers and switches.[J]. Nature Reviews Microbiology, 2012, 10(4):255-65.
    • [2]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.
    • [3]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.
    • [4]Breaker R R. RNA Switches Out in the Cold[J]. Molecular Cell, 2010, 37(1):1-2.
    • [5]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.