lldRO1-J23118-lldRO2

Collection of promoters regulated by LldR.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 78
Illegal NheI site found at 101
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Usage and Biology

Figure 1. Theoretical model for the dual function as repressor and activator of LldR.

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.

Characterization of the promoter

We wanted to know how the promoter would respond to the lactate presence and how important is the architecture of the promoter in this response. For this regard, we built several promoters. Part:BBa_K1847002, Part:BBa_K1847003, Part:BBa_K1847004, Part:BBa_K1847005, Part:BBa_K1847006, Part:BBa_K1847007, Part:BBa_K1847008, and Part:BBa_K1847009.

Genetic design for characterization with LldR

To test our 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 (Part:BBa_J23118) and a strong RBS (Part:BBa_B0034) in pSEVA371 backbone. According to literature [1], LldR will repress the promoter and by the addition of lactate the production of GFP will start, as the repressor will be removed.

Genetic design for characterization with LldP-LldP

The second step for our characterization was to add an L-lactate permease (LldP), which despite its name is an active transporter of L-lactate, D-lactate and glycolate. To understand the effect of LldP (L-lactatep permease) in our system, we tried the following conditions:

Plasmid with the promoter and sfGFP in medium copy number plasmid+ J23114-B0032-lldP-lldR (LldP and LldR are under the control of the same low promoter) in low copy number plasmid
Plasmid with the promoter and sfGFP in medium copy number plasmid + J23118-B0034-lldP-lldR (LldP and LldR are under the control of the same medium promoter) in low copy number plasmid

Plate reader

E. coli TOP10 strains were grown overnight in Lysogeny Broth (LB) containing kanamycin (50 µg/mL) and chloramphenicol (12.5 µg/mL) 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 in a Tecan Infinite M200 Pro Plate Reader. We calculated dose-response curves from the exponential phase of the bacteria using normalized fluorescence corrected by optical density.

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
K_M: half-maximum effective concentration (a representation of sensitivity) in µM
n: Hill coefficient (a representation of cooperativity), unitless

	J23118-B0034-lldR	J23118-B0034-lldP-lldR	J23114-B0032-lldP-lldR
Coefficient values	K_M=977.5 (812.6, 1142)	K_M=2361 (1051, 3671)	K_M=459.8 (121.5, 798)
	al = 1.1e+04 (fixed at bound)	al = 516.7 (-1522, 2555)	al = 1.775e+04 (1.619e+04, 1.93e+04)
	mu = 1.717e+04 (1.634e+04, 1.8e+04)	mu = 1.256e+04 (9078, 1.603e+04)	mu = 1.711e+04 (1.366e+04, 2.057e+04)
	n1 = 1.084 (0.8159, 1.352)	n1 = 0.7986 (0.3991, 1.198)	n1 = 0.7485 (0.3491, 1.148)
Goodness to fit	sse: 5.5485e+08	sse: 3.3911e+08	sse: 9.3519e+09
	rsquare: 0.9967	rsquare: 0.9894	rsquare: 0.9835
	fe: 8	dfe: 7	dfe: 7
	adjrsquare: 0.9959	adjrsquare: 0.9848	adjrsquare: 0.9764
	rmse: 8.3280e+03	rmse: 6.9602e+03	rmse: 3.6551e+04

Figure 2. Comparison of the fluorescence levels obtained using the lldR promoter alone and in combination with LldR and LldP-LldR. Mean fluorescence obtained with n=3 ± SD.

As can be seen in the picture above, when P_lldR GFP is alone, the fluorescence levels do not vary with lactate concentration. However, if P_lldR GFP is expressed in a system with constitutive expression of LldR, at low concentrations of lactate there is repression of the promoter, while at high concentrations there is activation. Activation is higher when there is constitutive expression of LldP-LldR (symbolized in the graph as LldPR).

This promoter has leakiness when it is expressed with LldP-LldR. Its ON/OFF ratio is smaller than the one in the natural promoter (see Table 1).

This promoter is one from our designed and tested synthetic promoters based on the wild-type P_lldR. In the following table, one can see the ON/OFF ratios and the K_M of the wild-type promoter and the three more prominent members of the library.

Table 1: ON/OFF ratio

	LldR	Additional LldP
	J23118-B0034-lldR	J23118-B0034-lldP-lldR	J23114-B0032-lldP-lldR
Part:BBa_K822000 (wild-type)	10.35	8.04	1.16
Part:BBa_K1847008	15.26	23.96	1.42	Increasing promoter strength
Part:BBa_K1847009	2.5	24.34	0.96
Part:BBa_K1847007	2.15	3.85	1.29

Table 2: K_M (µM)

	J23118-B0034-lldR	J23118-B0034-lldP-lldR	J23114-B0032-lldP-lldR
Part:BBa_K822000 (wild-type)	955	1930	720.2
Part:BBa_K1847008	1075	1751	337.5
Part:BBa_K1847009	977.5	2361	459.8
Part:BBa_K1847007	697.7	1977	1337

References

J Bacteriol. 2008 Apr; 190(8): 2997–3005.