Difference between revisions of "Part:BBa K2588000"

Latest revision as of 01:33, 18 October 2018

Glucose-repressed promoter MlcRE

MlcRE is a glucose-repressed promoter derived from Escherichia coli. In contrast to other commonly used inducible promoters that are repressed by glucose, like lacI promoter BBa_R0010, or pBAD promoter BBa_K206000, MlcRE does not rely on an activation signal, but is constitutively active unless repressed by glucose. It is especially useful in applications in which E. coli needs to respond to nutrition availability autonomously.

Usage and Biology

MlcRE can be cloned upstream of any gene of interest to activate transcription in absence of glucose.

Characterization

We cloned MlcRE upstream of BBa_E0840 to generate a GFP generator controlled by MlcRE, which we registered as BBa_K2588039. We grew E. coli DH5α cultures with pSB1C3-BBa_K2588039 to OD₆₀₀ of 0.1 in glucose-free LB media. Present GFP background was eliminated by incubation under high light for 10 min. Cells were inoculated with 0.125, 0.25, 0.5, 1 or 2 mg/mL glucose and incubated at 37°C for 2 hours. For comparison to other registry promoters, we employed InterLab postive control BBa_I20270 as GFP fluorescence standard. To account for the different ribosome binding sites used in BBa_K2588039 and BBa_I20270, GFP expression was normalized by RBS strength of BBa_B0030, and BBa_B0032, respectively. Untransformed E. coli DH5α served as negative control.

After 2h, GFP expression was measured on a TECAN Spectra Fluor plate reader, and fluorescence was normalized by OD₆₀₀ (Fig. 1).

Without glucose, MlcRE expression is comparable to InterLab positive control BBa_I20270, classifying it as strong promoter. MlcRE reaches saturation at 1 mg/mL glucose with a dynamic range of 4.3-fold induction between active and repressed state.

Sequence and Features