Difference between revisions of "Part:BBa K389421"

Revision as of 12:54, 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

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/B promoter element and downstream by the reporter K389004, a luciferase (Figure 1). 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

@@ Line 5: / Line 5: @@
 ===Usage and Biology===
+'''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.
+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/B promoter element and downstream by the reporter [https://parts.igem.org/Part:BBa_K389004 K389004], a luciferase (Figure 1). 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: