Latest revision as of 22:32, 27 September 2015

lldRO1-plac-lacO-lldRO2

Collection of promoters regulated by LldR and LacI.

Usage and Biology

Figure 1. Scheme of the activity of the double promoter.

The natural promoter of LldR (Part:BBa_K822000) consists of two operators (O1 and O2) and a promoter which is intercalated between the operators. It regulates the expression of the lldPRD operon, and it is involved in L-lactate metabolism. This promoter is repressed by a dimer of LldR, possibly by forming a DNA loop that does not allow the RNA polymerase to bind to the promoter. LldR can also have a function as an activator [1]. The repression of the promoter can be removed by lactate.

Our biobrick is a hybrid promoter repressed by LldR and LacI.

Characterization of the promoter

We wanted to know how the promoter would respond to the presence of lactate and/or IPTG, and how important is the architecture of the promoter in this response. For this regard, we built several promoters. Part:BBa_K1847010, Part:BBa_K1847011, and Part:BBa_K1847012. For characterization, gfp was cloned behind the hybrid promoter.

Experimental results

The promoter is responsive to both IPTG and lactate, being it more active when both are present in relatively high concentrations in the medium. A heat map of the fluorescence intensity can be seen in the Figure 3.

Figure 2. Expected result for the hybrid promoter

Figure 3. Dose-response of the hybrid promoter with 0 to 50 mM IPTG and 0 to 335 mM lactate. Fluorescence of GFP was measured in exponential phase and normalized by OD. Single measurements.

After the first experiment, new measurements were taken with the most promising concentrations (0-100 µM IPTG and 0-40 mM lactate), which can be seen in Figure 4.

Figure 4. A) Dose-response of the hybrid promoter in a narrower range of concentrations. B) Representation of the AND gate behavior of the hybrid promoter. Experiments are means with n=3±SD

Figure 5 is representing the AND gate behavior of the hybrid promoter. The leakiness for the lldR is much lower than for lacI, which could be due to the difficulties of access of the lacO by LacI when the DNA is looping [1].

Experimental Set-Up of hybrid promoter

Plasmids

To test our double hybrid we designed a plasmid containing the promoter with sfGFP in a medium copy plasmid and transformed it into Escherichia coli TOP10. Then, we added a second plasmid containing lldP and lldR with a medium strong promoter (Part:BBa_J23118) and a strong RBS (Part:BBa_B0034). Finally, we transformed another plasmid with LacI. LldR and LacI will repress the promoter. Upon addition of IPTG, LacI repression will be removed. Upon addition of lactate, LldR repression will be removed. Only when the two inducers are present GFP can get expressed.

Plate reader

E. coli TOP10 strains were grown overnight in Lysogeny Broth (LB) containing kanamycin (50 µg/mL), chloramphenicol (12.5 µg/mL) and ampicillin (50 µg/mL) as required at 37°C and 200 rpm. Cultures were diluted 1:50 in fresh LB with the corresponding antibiotic and transferred to a 96-well plate (200 µL/well). Cultures were grown for 90 min to arrive to exponential phase and then different concentrations of lactate from 0 to 335 mM and concentrations of IPTG from 0 to 50 mM were added. A blank of LB with the corresponding lactate concentration was done. During 7 h the absorbance at OD600 and fluorescence (excitation 488 nm and emission 530 nm) were measured with intervals of 7 min. The plate was always kept at 37°C. We calculated dose-response curves from the exponential phase of the bacteria.

After the first results were obtained, we selected the most promising concentrations of lactate and IPTG and repeated the measurement with the same conditions as before but with triplicates.

Sequence and Features