Revision as of 09:25, 10 October 2018

Cold-repressible RNA 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 cold-repressible RNA thermosensors can repress translation of downstream genes at low temperatures. The composite part is composed of promoter BBa_J23106, cold-repressible RNA thermosensor-4 BBa_K2541204, reporetr protein sfGFP BBa_K2541400 and terminator BBa_B0015.

Usage and Biology

Cold-repressible RNA thermosensor-4

RNA-based temperature sensing is common in bacteria that live in fluctuating environments. Most naturally-occurring RNA thermosensors have long sequences and complicated sencondary structure. Here, we designed short, 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

Green fluorescent protein (GFP) exhibits intrinsic fluorescence and is commonly used as a reporter gene in intact cells and organisms. Many mutants of the protein with either modified spectral properties, increased fluorescence intensity, or improved folding properties have been reported.

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 that folds well even when fused to poorly folded polypeptides. There is another superfolder GFP designed by Overkamp W et al at 2013, which is codon optimized for S. pneumoniae. It was be used in Escherichia coli by Segall-Shapiro T H et al at 2018.

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 stronger fluorescence intensity than superfolder GFP(BBa_I746916).

Conclusion

The composite part can be used as a measurement device for different cold-repressible RNA thermosensors. Wu use GoldenGate assembly to change differrnt thermosensors to measure their melting temperature.

Characterization

The thermosensor is constructed on the pSB1C3 vector by GoldenGate assembly. As shown below, the measurement device is composed of Anderson promotor (BBa_J23106), thermosensor (BBa_K2541204) and sfGFP (BBa_K2541400) and terminator (BBa_B0015). We measured the sfGFP expression to get the state of the cold-repressible RNA thermosensor at different temperatures.

As shown in the figure, the thermosensor is "off" at [ ]. 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