Difference between revisions of "Part:BBa K389422"

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===Usage and Biology===
 
===Usage and Biology===
  
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==Output-signal amplification by Sensitivity Tuner implementation==
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==Output-signal amplification by sensitivity tuner implementation==
  
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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]).
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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://2007.igem.org/Cambridge/Amplifier_project iGEM Team Cambridge, 2009]).
  
  
  
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'''Purpose of Sensitivity Tuner application'''
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'''Purpose of sensitivity tuner application'''
  
 
We presumed weak expression rates of our reporter luciferase indicated by pretesting the native system [https://parts.igem.org/Part:BBa_K389015 K389015]. For having a broader range of quantification for our prototype test system, an amplification device was implemented.  
 
We presumed weak expression rates of our reporter luciferase indicated by pretesting the native system [https://parts.igem.org/Part:BBa_K389015 K389015]. For having a broader range of quantification for our prototype test system, an amplification device was implemented.  
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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 ([https://parts.igem.org/Part:BBa_I746370 I746370]), 10 ([https://parts.igem.org/Part:BBa_I746370 I746380]) and 35 ([https://parts.igem.org/Part:BBa_I746370 I746390]) removed pBAD/araC promotor ([https://parts.igem.org/Part:BBa_I0500 I0500]) and GFP ([https://parts.igem.org/Part:BBa_E0040 E0040]) by self- designed primer PCR and replaced it upstream by a VirA/G response regulater [https://parts.igem.org/Part:BBa_K389015 K389015] and Virb promoter element [https://parts.igem.org/Part:BBa_K389003 K389003] and downstream by the reporter [https://parts.igem.org/Part:BBa_K389004 K389004], a luciferase (Figure 1). The RBS from the original Sensitivity Tuner for the phage activator was thereby removed. New RBS comes with Virb-promotor element [https://parts.igem.org/cgi/sequencing/one_blast.cgi?id=7888 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]).
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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 ([https://parts.igem.org/Part:BBa_I746370 I746370]), 10 ([https://parts.igem.org/Part:BBa_I746370 I746380]) and 35 ([https://parts.igem.org/Part:BBa_I746370 I746390]) removed pBAD/araC promoter ([https://parts.igem.org/Part:BBa_I0500 I0500]) and GFP ([https://parts.igem.org/Part:BBa_E0040 E0040]) by self- designed primer PCR and replaced it upstream by a VirA/G response regulater [https://parts.igem.org/Part:BBa_K389015 K389015] and ''virB''-promoter element [https://parts.igem.org/Part:BBa_K389003 K389003] and downstream by the reporter [https://parts.igem.org/Part:BBa_K389004 K389004], a luciferase (Figure 1). The RBS from the original sensitivity tuner for the phage activator was thereby removed. New RBS comes with ''virB''-promoter element [https://parts.igem.org/cgi/sequencing/one_blast.cgi?id=7888 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]).
  
  

Latest revision as of 16:17, 10 May 2013

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://2007.igem.org/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 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 original sensitivity tuner for the phage activator 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