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

RNA thermosensors

Heat-inducible RNA thermosensors

Heat-inducible RNA thermosensors are RNA-based genetic control systems that sense temperature changes. At low temperatures, the mRNA adopts a stem-loop conformation that masks the ribosome binding site [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 ribosome binding site to initiate translation. Whereas natural RNA thermosensors have a relatively complicated secondary structure with multiple stems, hairpin loops and bulges. The highly complex RNA secondary structures into which most naturally occurring RNA thermosensors can be folded has led to the hypothesis that RNA thermosensors may not function as simple on/off switches. Our team designed synthetic heat-inducible RNA thermosensors that are considerably simpler than naturally occurring thermosensors and can be exploited as convenient on/off switches of gene expression.

Heat-repressible RNA thermosensors

RNA-based temperature sensing is common in bacteria that live in fluctuating environments. Most naturally-occurring RNA thermosensors are heat-inducible, have long sequences, and function by sequestering the ribosome binding site in a stem-loop structure at lower temperatures. Here, we designed short, heat-repressible RNA 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. At elevated temperatures, the stem-loop unfolds, allowing for mRNA degradation and turning off expression. These short, modular heat-repressible RNA 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, the most widely studied of which are the Csp family of cold shock proteins in E. coli. CspA family is represented by 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 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 Shine–Dalgarno (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 Shine–Dalgarno (SD) sequence to block translation initiation region, which impedes translation. In our design, we deleted the conserved region called the cold box 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 increase base pairing or increase GC content, which may increase the temperature threshold for pseudoknot unfolding; we reduce base pairing or reduce GC content, which may cause the temperature threshold for pseudoknot unfolding to drop. Our team designed synthetic cold-inducible RNA thermosensors that are considerably simpler than naturally occurring cspA thermosensors and can be exploited as convenient on/off switches of gene expression.

Cold-repressible RNA thermosensors

Cold-repressible RNA thermosensors, which will form a stem-loop upstream Shine–Dalgarno (SD) sequence. These thermosensors contain a double-strand RNA cleavage site for RNase III, an enzyme native to Escherichia coli and many other organisms, in the 5' untranslated region of the target gene. 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. 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), which emission and excitation wavelength are similar to GFP, but its fluorescence intensity and folding speed are higher than GFP. Because of its advantages, our measurement device used sfGFP as the reporter protein. However, since we use Goldengate assembly this year, the existing sfGFP (BBa_I746916) in the registry contains a BbsI endonuclease cutting site. We designed a site-directed mutation of sfGFP (BBa_K2541401), made a double-base mutation to the BbsI recognition site without changing its amino acid sequence. BBa_K2541401 is completely suitable for Goldengate assembly, so we also call it sfGFP for Goldengate.

There's still one problem that we cannot predict the effect of the sfGFP after mutation. In addition, we found a codon optimized sfGFP for prokaryote, and called it sfGFP_optimism. Then we designed a composite part, which contains J23103, B0034 and sfGFP_optimism, and did experiment to compare it with sfGFP and sfGFP for goldengate. As the result shows, the sfGFP_optimism has higher fluorescence intensity than others, so we finally chose sfGFP_optimism as our reporter gene.

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 the sfGFP_optimism to the parts registry.

Basic parts content