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| | ===Applications of BBa_I746370=== | | ===Applications of BBa_I746370=== |
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| − | '''Purpose of Sensitivity Tuner application'''
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| − | 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/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]).
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| − | '''Characterization tests'''
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| − | 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 ([https://parts.igem.org/Part:BBa_K389015 K389015]; without tuning elements). Induction was done upon inoculation. Measuring point for amplification factor calculation was OD 1.0.
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| − | '''Results'''
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| − | Three sensitivity tuned Vir-Gen sensing systems were obtained: [https://parts.igem.org/Part:BBa_K389421 K389421], [https://parts.igem.org/Part:BBa_K389422 K389422] and [https://parts.igem.org/Part:BBa_K389423 K389423] distinguishing by the amplification level of luc transcription.
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| − | [[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.]]
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| − | The amplification factor was received by apply [https://parts.igem.org/Part:BBa_K389015 K389015] as reference. Amplification calculation was done by normalizing relative luminescence units emitted from luciferase per OD.
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| − | Output-signal amplification is in the induced contructs (red) [https://parts.igem.org/Part:BBa_K389422 K389422] and [https://parts.igem.org/Part:BBa_K389423 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 [https://parts.igem.org/Part:BBa_I746390 I746390]) shows the highest amplification rate of all tested Sensitivity Tuners. Our results indicate to higher amplification rate of [https://parts.igem.org/Part:BBa_K389421 K389421] than [https://parts.igem.org/Part:BBa_K389422 K389422] of 100 fold under induced conditions. The controls also show high basal transcription rates.
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| − | 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.
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| − | For further theory click [http://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Read_out_system Read out system]
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| − | 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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| | ===User Reviews=== | | ===User Reviews=== |
| | <!-- DON'T DELETE --><partinfo>BBa_I746370 StartReviews</partinfo> | | <!-- DON'T DELETE --><partinfo>BBa_I746370 StartReviews</partinfo> |
| − | <!-- Template for a user review
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| | {|width='80%' style='border:1px solid gray' | | {|width='80%' style='border:1px solid gray' |
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| − | <partinfo>BBa_I746370 AddReview number</partinfo> | + | <partinfo>BBa_I746370 AddReview 5</partinfo> |
| − | <I>Username</I> | + | <I>Bielefeld-Germany-2010</I> |
| | |width='60%' valign='top'| | | |width='60%' valign='top'| |
| − | Enter the review inofrmation here.
| + | We used and modified this part. Therefore we removed the arabinose promoter and replaced the GFP reporter with a firefly luciferase <partinfo>K389401</partinfo>. It can be shown, that the amplifation works. For Experience and further Data: <partinfo>K389421</partinfo> |
| | |}; | | |}; |
| | <!-- End of the user review template --> | | <!-- End of the user review template --> |
| | <!-- DON'T DELETE --><partinfo>BBa_I746370 EndReviews</partinfo> | | <!-- DON'T DELETE --><partinfo>BBa_I746370 EndReviews</partinfo> |
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