Revision as of 09:02, 10 October 2018

Heat-inducible RNA-based thermosensor measurement device

A RNA thermosensor that can be used for temperature sensitive post-transcriptional regulation which is based on the change of RNA sencondary structure. The heat-inducible RNA thermosensors can initiate translation of downstream genes at high temperatures. The composite part is composed of promoter BBa_J23104, heat-inducible RNA thermosensor-51 BBa_K2541051, reporetr protein sfGFP BBa_K2541400 and terminator BBa_B0015.

Usage and Biology

Heat-inducible RNA thermosensor-51

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.

sfGFP

The composite part can be used as a measurement device for different heat-inducible RNA thermosensors. Wu use goldengate assembly to change differrnt thermosensors to measure their melting temperature. sfGFP (BBa_K2541400) is BbsI restriction site free, so it can be used in goldengate assembly to achieve efficient and rapid assembly of gene fragments.

Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo, and many different variants are currently available to study bacterial cell biology. It exhibits intrinsic fluorescence and is commonly used as a reporter gene in intact cells and organisms [1]. Many mutants of the protein with either modified spectral properties, increased fluorescence intensity, or improved folding properties have been reported [2].

GFP often misfold when expressed as fusions with other proteins, while a robustly folded version of GFP, called superfolder GFP, was developed and described by Pédelacq et al at 2006 [3]that folds well even when fused to poorly folded polypeptides. Furthermore, sfGFP might be particularly suitable for gene expression studies, since the emergence of fluorescence closely matches induction of transcription. The superfolder GFP had been registered in iGEM BBa_I746916. Its codon usage is a compromise for optimum expression in E. coli and B. subtilis. There is another superfolder GFP designed by Overkamp W et al at 2013[4], which is codon optimized for S. pneumoniae. It was be used in Escherichia coli by Segall-Shapiro T H et al at 2018[5].

This year our team registered the superfolder GFP designed by Overkamp W et al with a BBa_K2541400 (called sfGFP). Compared to superfolder GFP(BBa_I746916), sfGFP (BBa_K2541400) is BbsI restriction site free, so it can be used in goldengate assembly to achieve efficient and rapid assembly of gene fragments. And sfGFP (BBa_K2541400) has x fold increased fluorescence intensity than superfolder GFP(BBa_I746916).

Characterization

The thermosensor is constructed on the pSB1C3 vector by goldengate assembly. As shown below, the measurement device is composed of Anderson promotor (BBa_J23104), heat-inducible RNA thermosensor-51 (BBa_K2541051), sfGFP (BBa_K2541400) and terminator (BBa_B0015). We measured the sfGFP expression to get the actual melting temperature of the heat-inducible RNA thermosensor.

As shown in the figure, the thermosensor melting temperature range is [ ]. Our data show that efficient RNA thermosensors can be built from a single small RNA stem-loop structure masking the ribosome binding site, thus providing useful RNA-based toolkit for the regulation of gene expression.

Sequence and Features