Part:BBa_K389422
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.
Usage and Biology
Output-signal amplification by Sensitivity Tuner implementation
Using an 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 pretesting 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 promotor (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 original Sensitivity Tuner for the phage activator war thereby removed. New RBS comes with Virb-promotor 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:
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE 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 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1632
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1768
Illegal SapI.rc site found at 6252
//chassis/prokaryote/ecoli
//classic/measurement/uncategorized
//function/reporter/light
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