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

RNA-based thermosensors

Heat-inducible RNA thermosensors

Heat-inducible RNA-based thermosensors are RNA-based genetic control systems that sense temperature changes. At low temperatures, the mRNA adopts a stem-loop that sequesters the ribosome binding site [Shine–Dalgarno (SD) sequence] within the 5′-untranslated region (5′-UTR). In this way, we could prevent ribosome binding and translation. At elevated temperature, the stem-loop melts, thereby making the ribosomes accessible to the ribosome binding site to initiate translation. There are some naturally-occurring RNA thermosensors, however, they have complicated secondary structures with multiple stems, loops and bulges, which makes them difficult to implement in engineered systems. 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 thermosensors

Most naturally-occurring RNA thermosensors are heat-inducible, which have long sequences and function by sequestering the ribosome binding site in a stem-loop structure at lower temperatures. Here, we also designed short heat-repressible RNA-based thermosensors. These thermosensors contain a single-strand RNA cleavage site for RNase E, an enzyme native to Escherichia coli and many other organisms, in the 5' untranslated region of the target gene. At low temperatures, the cleavage site is sequestered in a stem-loop and gene expression is unobstructed. Whereas at elevated temperatures, the stem-loop unfolds, and the mRNA is degradated and gene expression is turned off. These short, modular heat-repressible RNA-based thermosensors can be applied to the construction of complex genetic circuits, facilitating rational reprogramming of cellular processes for synthetic biology applications.

Cold-inducible RNA thermosensors

There are multiple families of cold-inducible proteins in prokaryotes, in which the most widely studied one are the CspA, which has been quite extensively investigated. 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 mRNA. At low temperatures (<20℃), the 5’UTR of CspA mRNA can form an advanced structure called pseudoknot. Then the pseudoknot conformation exposes the Shine–Dalgarno (SD) sequence and it is beneficial to recruit ribosomes and somewhat less susceptible to degradation. At normal temperatures, due to thermodynamic instability, pseudoknot unfolds. As a result of that, 5’UTR of CspA forms a secondary structure, which sequesters Shine–Dalgarno (SD) sequence and further block translation initiation region and impedes translation. In our project, we have deleted the conserved region called the cold box located upstream of the 5'UTR of CspA mRNA, so that the expression of CspA is not regulated by its own negative feedback. The pseudoknot in the CspA mRNA contains four sets of base pairings, and its stability is temperature-regulated. We increased the number of base pairing or increased the GC content of CspA gene, which may increase the temperature threshold for pseudoknot unfolding. In addition, we also reduced the number of base pairing or reduced the GC content, which could make the temperature threshold for pseudoknot unfolding drop down. Hence, our team has successfully designed synthetic cold-inducible RNA thermosensors that are considerably simpler than naturally occurring CspA thermosensors and those cold-inducible thermosensors can be exploited as convenient on/off switches of gene expression.

Cold-repressible RNA thermosensors

These thermosensors contain a double-stranded (ds) RNA cleavage site for RNase III, an ribonuclease native to Escherichia coli, which could cleave dsRNA in a highly site-specific manner. This cleavage site was inserted in the 5' untranslated region of the target gene. At low temperatures, the mRNA stem-loop is stable and the RNase III cleavage site is exposed, leading to the degradation of the mRNA. At elevated temperature, the stem-loop will unfold and gene translation will occur unhinder. These short, modular cold-repressible RNA thermosensors can be applied to the construction of complex genetic circuits, facilitating rational reprogramming of cellular processes for synthetic biology applications.

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 . However, the existing sfGFP (BBa_I746916) in the registry contains a BbsI endonuclease cleavage site, which is needed in Golden Gate assembly system in our program. 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 also called it sfGFP for Golden Gate assembly.

We also performed a codon optimization sfGFP for prokaryote, and named it sfGFP_optimism. Then we designed a composite part, which contains Anderson Promoter J23104, RBS B0034 and sfGFP_optimism. We did experiments to compare it with sfGFP and sfGFP for Golden Gate assembly. 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 Goldengate 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

References

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• [2]Kortmann J, Narberhaus F. Bacterial RNA thermometers: molecular zippers and switches.[J]. Nature Reviews Microbiology, 2012, 10(4):255-65.
• [3]Neupert J, Bock R. Designing and using synthetic RNA thermometers for temperature-controlled gene expression in bacteria. Nature Protocols, 2009, 4(9):1262-73.
• [4]Sen S, Apurva D, Satija R, et al. Design of a Toolbox of RNA Thermometers[J]. Acs Synthetic Biology, 2017, 6(8).
• [5]Hoynes-O'Connor A, Hinman K, Kirchner L, et al. De novo design of heat-repressible RNA thermosensors in E. coli[J]. Nucleic Acids Research, 2015, 43(12):6166-6179.
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• [7]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.
• [8]Breaker R R. RNA Switches Out in the Cold[J]. Molecular Cell, 2010, 37(1):1-2.
• [9]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.