Part:BBa_K598011

pBAD+TPP Down-regulated Hammerhead Ribozyme 2.5 with Native RBS+E0040+B0015

This is a basic part that consists of pBAD promoter, thiamine pyrophosphate (TPP) ligand responsive hammerhead ribozyme numbered 2.5 with native RBS (AAGGAGAT), BBa_E0040 and BBa_B0015.(fig.1)

Figure 1 Construction of pBAD+TPP Down-regulated Hammerhead Ribozyme 2.5 with Native RBS+E0040+B0015. This part consists of pBAD promoter, TPP ribozyme 2.5 with native RBS (AAGGAGAT), BBa_E0040 and BBa_B0015.It is obtained by PCR from inactive TPP-HHAz 2.5 [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 inhibit gene expression when induced by 1mM arabinose sensitively. The working curve is shown in fig.2. The inhibition ratio is the value of fluorescence intensity compared to that of without TPP.

Figure 2Working curve of BBa_K598011. The inhibition 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 arabinose.

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
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 125
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 65
  Illegal BamHI site found at 258
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 931