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−	<h1>BBa_K3245003:</h1>	+	<h1> luxpR-fus100</h1>
−	<p>BBa_K3245003（J23106-B0034-C0040-B0015-R0040-B0034-K3245008-B0014-B0015） is one of a series of composite parts designed for characterizing R0040.</p>	+	<p>This hybrid promoter has higher expression level when induced by luxR-AHL complex triggered by quorum sensing ( QS ) in some Gram-negative bacteria, meanwhile it’s leakage is also higher when not induced.</p>
	<h2>Usage and biology:</h2>		<h2>Usage and biology:</h2>
−	<p>This part is one of a collection of parts designed for characterizing ptetR (R0040). This part constantly expresses tetR(C0040) at middle-high level strength to inhibit the downstream ptetR from expressing sfGFP. When induced by aTc/tet, this part shows a leaping induction curve. To use this part, simply clone it into a middle/high copy plasmid vector, transfer into E.coli K-12, incubate overnight, and induce with aTc after proper dilution. </p>	+	<p>This fused promoter is designed for the situation that QS effect and high expression level are required at the same time in a circuit if leakage is acceptable to some extent. We applied this enhanced promoter to improve lacZ expression when bacteria reached a high density. </p>
	<h2>Design:</h2>		<h2>Design:</h2>
−	<p>Since our project involves tetR-ptetR double plasmid expression system (Fig.1), it is essential for us to characterize ptetR by measuring the level of tetR expression required for total inhibition. To achieve this, we designed 3 different tetR-ptetR expression systems (BBa_K3245003, BBa_K3245012, BBa_K3245011), among which K3245003 expresses the highest amount of tetR (J23106-B0034). </p>	+	[[File:T--Fudan--Part50.png|700px]]
−	<p>In order not to let the promoter of antibiotics and J23106 affect the process of characterization, we inserted 2 B0015s into our expression system, one at the upstream of R0040, and the other at the downstream of sfGFP. </p>	+	<p>As the figure shows, we substituted the -35 to -10 region of the original promoter luxpR with the one of J23100, a strong constitutive promoter. This region is rarely concerned as it’s rather conservative in promoters with the same function, but it has crucial structural effect on σ factor binding and other events in transcription regulation. Fortunately the change proved to be effective on adjusting the behavior of the regulatory promoter.</p>
−	[[File:T--Fudan--Part1.png|700px]]	+
	<h2>Characterization:</h2>		<h2>Characterization:</h2>
−	<p>In order to characterize R0040, we cloned K3245003 into p15A, a vector with middle copy number (15-20). We then transferred p15A-K3245003 into E.coli DH10B and incubated it overnight. The culture was diluted with LB to 1/500 before induction (see team Fudan_protocol for more detail). Due to lack of access to aTc, we use tetracycline (tet) to induce R0040. Our results are shown as follows: </p>	+	<p>Measurement Protocol</p>
		+	<p>1. Transform a control plasmid containing luxI and luxR ( BBa_K3245002) with pUC ori ( high copy number ) and an effect plasmid containing luxpR-fus100 and GFP behind the promoter with p15A ori (medium copy number ) into DH10B. </p>
		+	<p>2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 3 ml LB with ampicillin at 100 μg/ml. Incubate overnight at 37℃ in a shaker.</p>
		+	<p>3. Measure and keep all groups OD600 reach 1. Inoculate each group with with 1/5000 concentration. Incubate 12 hours at 37℃ in a shaker.</p>
		+	<p>4. Add 100 µl bacteria culture medium into each well of a 96-well plate. One well of LB as blank, one group of wild type DH10B as control.</p>
		+	<p>5. Measure OD600 and fluorescence continuously every 30 minutes with a microplate reader.</p>
		+	[[File:T--Fudan--Part51.png|400px]]
		+	<p><strong>Fig.1</strong> This figure used OD600 as X-axis unit. The MEFL/particle changes as the OD goes up after dilution. The downward curve before OD600 < 0.1 is due to the parental fluorescence and the dilution effect after proliferation. Data points are shown in the figure for fitting analysis. Here gives the parameters of the fitting equation.
		+	<h2>Reference</h2>
		+	Antunes, L. C., et al. "A mutational analysis defines Vibrio fischeri LuxR binding sites." Journal of Bacteriology 190.13(2008):4392-4397.
		+	de Boer HA, Comstock LJ, Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983;80(1):21–25. doi:10.1073/pnas.80.1.21

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Latest revision as of 03:56, 22 October 2019

luxpR-fus100

This hybrid promoter has higher expression level when induced by luxR-AHL complex triggered by quorum sensing ( QS ) in some Gram-negative bacteria, meanwhile it’s leakage is also higher when not induced.

Usage and biology:

This fused promoter is designed for the situation that QS effect and high expression level are required at the same time in a circuit if leakage is acceptable to some extent. We applied this enhanced promoter to improve lacZ expression when bacteria reached a high density.

Design:

As the figure shows, we substituted the -35 to -10 region of the original promoter luxpR with the one of J23100, a strong constitutive promoter. This region is rarely concerned as it’s rather conservative in promoters with the same function, but it has crucial structural effect on σ factor binding and other events in transcription regulation. Fortunately the change proved to be effective on adjusting the behavior of the regulatory promoter.

Characterization:

Measurement Protocol

1. Transform a control plasmid containing luxI and luxR ( BBa_K3245002) with pUC ori ( high copy number ) and an effect plasmid containing luxpR-fus100 and GFP behind the promoter with p15A ori (medium copy number ) into DH10B.

2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 3 ml LB with ampicillin at 100 μg/ml. Incubate overnight at 37℃ in a shaker.

3. Measure and keep all groups OD600 reach 1. Inoculate each group with with 1/5000 concentration. Incubate 12 hours at 37℃ in a shaker.

4. Add 100 µl bacteria culture medium into each well of a 96-well plate. One well of LB as blank, one group of wild type DH10B as control.

5. Measure OD600 and fluorescence continuously every 30 minutes with a microplate reader.

Fig.1 This figure used OD600 as X-axis unit. The MEFL/particle changes as the OD goes up after dilution. The downward curve before OD600 < 0.1 is due to the parental fluorescence and the dilution effect after proliferation. Data points are shown in the figure for fitting analysis. Here gives the parameters of the fitting equation.

Reference

Antunes, L. C., et al. "A mutational analysis defines Vibrio fischeri LuxR binding sites." Journal of Bacteriology 190.13(2008):4392-4397.

de Boer HA, Comstock LJ, Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983;80(1):21–25. doi:10.1073/pnas.80.1.21