Difference between revisions of "Part:BBa K389421"
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==Output-signal amplification by sensitivity tuner implementation== | ==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 ( | + | 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://2007.igem.org/Cambridge/Amplifier_project iGEM Team Cambridge, 2009]). |
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'''Purpose of Sensitivity Tuner application''' | '''Purpose of Sensitivity Tuner application''' | ||
| − | We presumed weak expression rates of our reporter luciferase indicated by | + | We presumed weak expression rates of our reporter luciferase indicated by pre-testing 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. |
| − | 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 | + | 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 phage activator from the original sensitivity tuner 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 23:27, 27 October 2010
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 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://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 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:
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