Part:BBa_K598015

T7-promoter+TPP Up-regulated Hammerhead Ribozyme 1.20 with Native RBS+GFP+T7-terminator

This is a basic part that consists of T7 promoter, thiamine pyrophosphate (TPP) ligand responsive hammerhead ribozyme numbered 1.20 with native RBS (AAGGAGAT), BBa_E0040 and BBa_B0015. (fig.1)

Figure 1 Construction of T7-promoter+TPP Up-regulated Hammerhead Ribozyme 1.20 with Native RBS+GFP+T7-terminator. This part consists of T7 promoter, TPP ribozyme 1.20 with native RBS (AAGGAGAT), BBa_E0040 and BBa_B0015.It is obtained by PCR from TPP-HHAz 1.20 [1] which is kindly provided by Markus Wieland et al., and then inserted into pSB1C3 through standard assembly.

This device can respond to different TPP concentration and activate gene expression when induced by 1mM IPTG sensitively. The working curve is shown in fig.2. The activation ratio is the value of fluorescence intensity compared to that of without TPP.

Figure 2Working curve of BBa_K598015. The activation ratio is fluorescence intensity under given TPP concentrations compared to that of without TPP. Constructed plasmids were transformed into E. coli DH5acells and characterized in M9 medium with a TPP concentration gradient of 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3 uM with being induced by 1mM IPTG.

Reference

[1] Markus Wieland, Armin Benz, Benedikt Klauser, and Jörg S. Hartig. (2009). Artificial Ribozyme Switches Containing Natural Riboswitch Aptamer Domains. Angew. Chem. 121, 2753-2756

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 171
  Illegal BamHI site found at 984
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

For the full characterisation of the device, please refer to BBa_K598004