Team:Strasbourg/Contribution

iGEM

iGEM registry represents a huge database, containing many of genetically engineered constructs, performed by iGEM teams during the last 15 years of the competition. These previously produced parts can be used by the iGEM community to optimize their experimental processes. Unfortunately, considering part of present items was not characterized experimentally before sending, its functions need to be confirmed before use. This year iGEM Foundation chooses "part characterization" as a bronze medal criteria, which is a better solution to extend the registry experimental database and increase the reliability of its parts.

iGEM Strasbourg team has access to fluorescence real-time plate reader (PheraStar Plus BM6 LABTECH), which is a good opportunity to obtain high fidelity results on kinetics of inducible promoters. Between the different parts sent by the iGEM Foundation for characterization, our team chose BBa_K731520 composite part. BBa_K731520 composite part represents LacI-LacIQ promoter (BBa_K731300) induction by measuring GFP expression (BBa_E0040). We 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, characterization Lab book.

Figure 1. Kinetics of LacI-LacIQ promoter (BBa_K731300) induction measured by GFP fluorescence (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 blank. Bacterial cells not expressing GFP were used as the negative control. n = 3.

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

Figure 2. GFP expression levels depending on 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.