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Part:BBa_I746370:Experience

Designed by: Stefan Milde   Group: iGEM07_Cambridge   (2007-09-26)
Revision as of 12:41, 27 October 2010 by Johnny (Talk | contribs) (→‎Applications of BBa_I746370)

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Applications of BBa_I746370

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]).


Characterization tests

Cultivation was done by induction with Acetosyringone at 50 µM. Controls were not induced Sensitivity Tuner devices as well as induced and not induced nativ system (K389015; without tuning elements). Induction was done upon inoculation. Measuring point for amplification factor calculation was OD 1.0.


Results

Three sensitivity tuned Vir-Gen sensing systems were obtained: K389421, K389422 and K389423 distinguishing by the amplification level of luc transcription.

[[Image:Bielefeld amp factor.png|400px|thumb|center| Figure 1: Amplification factor of induced, 50 µM Acetosyringone (red) and not induced (green) modified Sensitivity Tuner K389421, [K389422 and [K389423, Standard deviation shown.]]

The amplification factor was received by apply K389015 as reference. Amplification calculation was done by normalizing relative luminescence units emitted from luciferase per OD. Output-signal amplification is in the induced contructs (red) K389422 and K389423 100 and respectively 200 fold higher than in not induced controls (green). An exception is K389422 were induced and not indiced system revealed analog results. Corresponding to data of iGEM Team, Cambridge 2009, K389423 (originated from I746390) shows the highest amplification rate of all tested Sensitivity Tuners. Our results indicate to higher amplification rate of K389421 than K389422 of 100 fold under induced conditions. The controls also show high basal transcription rates.

Because there is small difference in induced and not induced system visible and basal transcription rates are high, we assume that the sensitivity tuning constructs are not well applicable for luciferase measurements.

For further theory click [http://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Read_out_system Read out system]



For test results click [http://2010.igem.org/Team:Bielefeld-Germany/Results/Tests#BBa_K389421.2C_BBa_K389422.2C_BBa_K389423:_Sensitivity_Tuner_amlified_Vir-test_system Sensitivity Tuner amlified Vir-test system]

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