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From 2010.igem.org

Characterization of <partinfo>K389421</partinfo>, <partinfo>K389422</partinfo>, <partinfo>K389423</partinfo>

By self designed PCR-Primer we excluded terminal GFP and the initial promoter pBAD/araC, for replacing our own VirB promotor and reporter gene luc (luciferase). Primers were designed for sensitivity tuner I746370, I746380 and I746390 so that standard assembly would be possible (Primer Design). Assembling of PCR-products took place by Silver Assembly.

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. (Protocols)

Results

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

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.

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