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−	<div style="margin:auto;width:880px">	+	[[File:ETH15_CombinedPromoterLactatelacUV5lldr.png|500px|center|Comparison of the fluorescence levels obtained using the wild-type promoter alone and in combination with LldR and LldP-LldR]]
−	<ul>	+	In the graph we can see that the promoter is more sensitive when it has LldP.
−	<li class="graphthumb" style="display:inline-block;width:33%">[[Image:ETH15_P74_LldR.png|thumb|260px|Dose-response curve for our promoter with J23118-B0034-lldR]]</li>	+
−	<li class="graphthumb" style="display:inline-block;width:33%">[[Image:P74_LldPRlow.png|thumb|260px|Dose-response curve for our promoter with J23114-B0032-lldP-lldR]]</li>	+
−	<li class="graphthumb" style="display:inline-block;width:33%">[[Image:ETH15_P74_LldPRhi.png|thumb|260px|Dose-response curve for our promoter with J23118-B0032-lldP-lldR]]</li>	+
−	</ul>	+
−		+
−	As you can see from the graphs, activation with only LldR is lower than with LldP-LldR. Also, the sample with LldP-LldR with a stronger promoter achieves higher fluorescence levels. We believe that this is due to the fact that more molecules of LldP are produced and therefore more lactate can enter inside the cell. Just as a qualitative example, J23118-B0034-lldR has half of the maximum fluorescence than J23118-B0034-lldP-lldR.	+
−	When comparing J23114-B0034-lldP-lldR and J23118-B0034-lldP-lldR, we can see that J23114-B0034-lldP-lldR has more leakiness, which we think is due to its low expression, and therefore it cannot repress the gene properly.	+
−		+
	<h1>References</h1>		<h1>References</h1>
	<ol>		<ol>

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Revision as of 18:11, 17 September 2015

lldRO1-placUV5-lacO-lldRO2

Promoter regulated by LldR and LacI.

Sequence and Features

Usage and Biology

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 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.

Experimental Set-Up for LldR response

Plasmids

To test our double promoter 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 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 (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.

Experimental Set-Up for lldR-lldP response

Plasmids

After the initial experiment with LldR, an L-lactate permease (LldP) was added to the plasmid to explore the possibilities when lactate import rates increase.

Plate reader

The same set-up as for LldR is applied.

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. A medium copy plasmid containing gfp activated by our synthetic promoter was always present in the cell. We used two different promoters in front of the lldRP operon, either medium strong (Part:BBa_j23118) or weak ([[]Part:BBa_J23114]).

FInally we combined the medium copy plasmid with gfp, the plasmid with lldP-lldR and lacI.

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

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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

	J23118-B0034-lldR	J23118-B0034-lldP-lldR	J23114-B0032-lldP-lldR
Coefficient values	K=491.7 (91.34, 892)	K=2442 (511.1, 4373)	K=457.7 (271.7, 643.8)
	al = 2220 (931.5, 3509)	al = 2396 (769.3, 4023)	al = 1.32e+04 (fixed at bound)
	mu = 8758 (7552, 9964)	mu = 1.57e+04 (9270, 2.212e+04)	mu = 6942 (5920, 7963)
	n1 = 0.7 (fixed at bound)	n1 = 1.156 (0.4928, 1.82)	n1 = 1.569 (-0.05875, 3.196)
Goodness to fit	sse:5.0244e+08	sse: 1.2664e+09	sse: 1.2386e+09
	rsquare: 0.9767	rsquare: 0.9852	rsquare: 0.9569
	dfe: 8	dfe: 7	dfe: 8
	adjrsquare: 0.9708	adjrsquare: 0.9788	adjrsquare: 0.9461
	rmse: 7.9250e+03	rmse: 1.3450e+04	rmse: 1.2443e+04

In the graph we can see that the promoter is more sensitive when it has LldP.

References

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

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