CHARACTERIZATION – T7 promoter in an existing part

The existing part: Part:BBa_K3431048

We retransformed E. coli BL21 with the plasmid of T7-RBS-RFP-Tr/pSB1C3 (BBa_K3431048). Red colors were clearly seen in LB agar plate and LB liquid culture containing 1mM of IPTG compared to non-IPTG as controls after an overnight culture (Fig. 1).

The transformed E. coli BL21 runs in a kinetic mode for 24 hr in the LB broth with or without IPTG. The RFP levels were read by Synergy H1 Hybrid Multi-Mode Reader - BioTek Instruments (Agilent Technologies, Inc.) at an excitation and emission wavelength of 584 nm and 607 nm, respectively. Regardless of some interfering background values in LB media were detected in the first 3 hr, it’s clear that the T7 promoter activities began to be induced around 2 hr and became significantly strong to a level of 1.5-fold induction compared to the controls without IPTG at 12 hr (Fig. 2). The data were consistent with the results seen by naked eyes (Fig. 1).

SUMMARY

We transformed E. coli BL21 with Part:BBa_K3431048 and test on IPTG induction assay. We observed the result (i.e., RFP expression) on LB agar plate and liquid culture in LB broth supplemented with 34ug/ml of chloramphenicol. Furthermore, we picked up 2 colonies from the plate and run them with or without IPTG in a 18-hour kinetic mode by a microplate reader at 37°C. The data clearly showed the part was functional in the presence of IPTG in a time-dependent manner. And we well documented on the Part's Main Page on the Registry.