Part:BBa_K389421

VirA/G signaling system + sensitivity tuner + luciferase

This part combines a complete VirA/G signaling system with a sensitivity tuner before a luciferase gene under the control of a vir promoter. The sensitivity tuner enhances a signal from a promoter so it enhances the readout luciferase.

Output-signal amplification by sensitivity tuner implementation

Using a standard, inducible promoter/reporter system, often results in weak reporter expression and so on in difficulties in quantification. An amplification of transcription rate of desired genes can be realized using so called sensitivity tuner devices. Amplification takes place as promoter induction upregulates a phage activator, which binds to a phage promoter upstream of a reporter. As result a PoPs input (inducer) generate a PoPs output at higher signal. PoPs is equivalent to the flow of RNA polymerase molecules along DNA ([http://jb.asm.org/cgi/content/abstract/178/19/5668 Julien and Calendar, 1996]),([http://parts.mit.edu/igem07/index.php/Cambridge/Amplifier_project iGEM Team Cambridge, 2009]).

Purpose of Sensitivity Tuner application

We presumed weak expression rates of our reporter luciferase indicated by pre-testing the native system K389015. For having a broader range of quantification for our prototype test system, an amplification device was implemented. For amplifying the output signal of luciferase induced by acetosyringone, three sensitivity tuner distinguished by the amplification factor were combined with our detection system. To modify the sensitivity tuner for our purpose we took BioBricks with amplification factors from 15 (I746370), 10 (I746380) and 35 (I746390) removed pBAD/araC promoter (I0500) and GFP (E0040) by self- designed primer PCR and replaced it upstream by a VirA/G response regulater K389015 and Virb promoter element K389003 and downstream by the reporter K389004, a luciferase (Figure 1). The RBS from the phage activator from the original sensitivity tuner was thereby removed. New RBS comes with VirB-promoter element K389003. The benefits of luciferase reporter instead of GFP are a broader range of measurement, higher sensitivity and low half-live making cinetic tests possible ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4F031H9-30&_user=10&_coverDate=01%2F31%2F1989&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_searchStrId=1514624813&_rerunOrigin=google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=ee400628b119490fcdc44ccdd856c4e8&searchtype=a Williams et al.1989]).

To see, how this BioBrick works, watch the following animation:

Animation of the functionality of the BioBricks BBa_K389421, BBa_K389422 and BBa_K389423.

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
  Illegal NheI site found at 647
  Illegal NheI site found at 2581
  Illegal NheI site found at 2604
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1632
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 5002
  Illegal NgoMIV site found at 6346
  Illegal NgoMIV site found at 6367
  Illegal AgeI site found at 4525
  Illegal AgeI site found at 4637
  Illegal AgeI site found at 4839
  Illegal AgeI site found at 6070
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1768
  Illegal SapI.rc site found at 6252