Revision as of 05:29, 10 October 2018

Cold-inducible 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 advanced structure. The cold-inducible RNA thermosensors can initiate translation of downstream genes at low temperatures. The composite part is composed of promoter BBa_J23100, cold-inducible RNA thermosensor-1 BBa_K2541301, reporetr protein sfGFP BBa_K2541400 and terminator BBa_B0015.

Usage and Biology

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.

The composite part can be used as a measurement device for different cold-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.

Characterization

The thermosensor is constructed on the pSB1C3 vector by goldengate assembly. As shown below, the measurement device is composed of Anderson promotor (BBa_J23100), thermosensor (BBa_K2541301) and sfGFP (BBa_K2541400) and terminator (BBa_B0015). We measured the sfGFP expression to get the actual melting temperature of the cold-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 RNA advanced structure masking the ribosome binding site, thus providing useful RNA-based toolkit for the regulation of gene expression.

Sequence and Features