Revision as of 13:44, 16 September 2015

lldRO1-placUV5-lacO-lldRO2

Promoter regulated by LldR and LacI.

Sequence and Features

Usage and Biology

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.

The lac operator is repressed by LacI. This repression can be removed by allolactose or by IPTG.

Characterization of the promoter

We wanted to know how the promoter would respond to the lactate presence and to 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.

Experimental Set-Up

Plasmids

To test our double promoter we designed a plasmid containing the promoter with sfGFP in a medium copy plasmid pSEVA261 and transformed it into Escherichia coli TOP10. Then, we added a second plasmid containing lldR with a medium strong promoter (BBa_J23118) and a strong RBS (BBa_B0034) in pSEVA371 backbone. Finally, we transformed another plasmid with LacI. We expect LldR and LacI to 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.

We tried another set-up in which we added LldP into our LldR plasmid. LldP is an L-lactate permease which import L-lactate into the cell. In summary, we tried our system in three different conditions:

• Plasmid with the double promoter and sfGFP in pSEVA261 + J23118-B0034-lldR in pSEVA371
• Plasmid with the double promoter and sfGFP in pSEVA261 + J23114-B0032-lldP-lldR (LldP and LldR are under the control of the same promoter) in pSEVA371
• Plasmid with the double promoter and sfGFP in pSEVA261 + J23118-B0032-lldP-lldR (LldP and LldR are under the control of the same promoter) in pSEVA371
• Plasmid with the double promoter and sfGFP in pSEVA261 + J23118-B0032-lldP-lldR (LldP and LldR are under the control of the same promoter) in pSEVA371 + lacI

Plate reader

E. coli TOP10 strains were grown overnight in Lysogeny Broth (LB) containing kanamycin (1 µ/mL), chloramphenicol (0.36 µ/mL) and ampicillin (1 µ/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 were added. Samples were always made in triplicates and 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.

Modeling

Each experimental data set was fitted to a Hill function using the Least Absolute Residual method (Fitting Toolbox in MatLab).

Where:

• al: leakiness in µM/min
• mu: constant in µM/min
• Km: half-maximum effective concentration (a representation of sensitivity) in µM
• n: Hill coefficient (a representation of cooperativity), unitless