Part:BBa_K2541408

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

Cold-inducible RNA thermosensor-1

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.

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-inducible 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_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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 639
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]