Composite

Part:BBa_K731520:Experience

Designed by: Francesco Guzzonato   Group: iGEM12_TRENTO   (2012-08-22)

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Caracterization of BBa_K731520

iGEM Strasbourg team has access to fluorescence real-time plate reader (PheraStar Plus BM6 LABTECH), which is a good opportunity for obtaining high fidelity results on kinetics of inducible promoters. Our team chooses BBa_K731520 composite part for measured GFP expression rates under four different conditions : 0 μM, 20 μM, 100 μM and 500 μM of IPTG. Non-expressing GFP XL1 Blue bacteria were used as the negative control.

For detailed experimental protocol see iGEM Strasbourg 2019, (Lab work, Characterization ).

Figure 1. iGEM Strasbourg team establish kinetics of LacI-LacIQ promoter (BBa_K731300) induction by measuring GFP expression (BBa_E0040). Three different clones of XL1 Blue cells containing BBa_K731520 part were incubated overnight under rotation at 37° in LB media with supplementary 2% D-Glucose. After 18h the OD600 was adjust to OD = 0,1. The cells were incubated in presence of a different concentration of IPTG (0 μM, 20 μM, 100 μM and 500 μM) and PheraStar Plus BM6 LABTECH was used for real-time GFP measure. LB media was used as blanc. Bacterial cells not expressing GFP was used as the negative control. n = 3.

Using data from BBa_K731520 characterization we calculate the start point of GFP expression levels depending on IPTG concentration (Figure 2). These results could help the future teams to better choose experimental conditions using BBa_K731520 part.

Figure 2. GFP expression levels depending to IPTG concentration. The basic levels of GFP were calculated from BBa_K731520 characterization, n = 3. Equation applicated for establish this relation is y = 0,7206x + 7410,6.