Difference between revisions of "Part:BBa K518013"

Latest revision as of 20:17, 5 October 2011

sulA promoter evaluation device

Microbes including Escherichia coli are known to respond to various DNA-injuring stress (ionizing radiation, ultraviolet radiation, peroxides etc...), altering their gene expression patterns. This response, known as the "SOS response", is induced by a regulatory protein called RecA when it binds to single-strand DNA. The DNA-RecA complex promotes the degradation of LexA, a common repressor of SOS genes.

SulA is responsible for stress-induced halt of cell division. The promoter of sulA, sulAp, is induced by various stress factors, including ultraviolet irradiation.

We provide a sulAp evaluation device to make it easy to measure the varying expression of sulAp in various "stressful" conditions. Employing our dual luciferase assay kit, quantitative measurement and comparison of Relative Promoter Unit (RPU) can be achieved.

Measurement1: Characterization of the dual luciferase assay kit (BBa_K518002)

Calibration for Firefly Luciferase

First, we carried out a calibration for Photynus pyralis (Firefly) luciferase using standard reagents of enzyme and D-luciferin. Reagents were granted from Berthold Japan (Tokyo, Japan). Luminescence was measured using a luminometer GENE LIGHT 200 from MICROTEC, Co., Ltd. (Chiba, Japan).

The result indicates that under 1000 luciferase molecules can be detected, and that luciferase amounts can be well estimated by a simple linear regression in a logarithmically seven-digit range (from 10^(-20) to 10^(-13) mol). Again, we can easily get the numerical information of how many molecules exist, not just the arbitrary luminescence units.

Since we can estimate the number of cells in a test tube from various data including OD600 value, we are able to estimate how many luciferase molecules are translated in a single bacterium on average.

Test for BBa_K518002

We first performed a test for BBa_K518002 using BBa_J23119 and BBa_R0011. We successfully verified the IPTG-dependence of BBa_R0011, and at the same time, the potency of BBa_K518002 as an evaluation tool. For experimental and analytical details, see [http://2011.igem.org/Team:UT-Tokyo our page].

<Fig.1: Time-dependent luminescence measurement of firefly luciferase. The first graph shows the raw data obtained by triplicate experiments. Data is presented as mean ± S.D.. The second graph is a model fitting according to the enzymatic kinetics theory.>

<Fig.2: Time-dependent luminescence measurement of renilla luciferase.>

<Fig.3: Demonstration of promoter evaluation. Relative Promoter Unit (RPU) is calculated as: (maximum of firefly luminescence) / (maximum of renilla luminescence). Data is obtained from triplicate experiments. Data is expressed as mean±S.D..>

Measurement2: sulAp evaluation

The expression levels of sulAp were evaluated before and after UV induction. We evaluated this using both a recA(-) (JM109) and a recA(+) (BL21) strain. RecA is known to be necessary for releasing sulAp from repression. We successfully demonstrated a significant alteration of expression in recA(+) strain only after UV irradiation. The expression level of BBa_J23119, a constitutive E. coli promoter which is often used as a comparison, was simultaneously presented as a comparison. Data is expressed as mean ± S.D.. Data is obtained from the average of three independent experiments.

<Figure: UV-induced expression levels of sulAp.>

For experimental details, see [http://2011.igem.org/Team:UT-Tokyo our page].

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1850
Illegal NheI site found at 1873
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 2452
Illegal SapI.rc site found at 887

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K518013 short</partinfo>
-Microbes including Escherichia coli are known to respond to various DNA-injuring stress (ionizing radiation, ultraviolet radiation, peroxides etc...), altering their gene expressions. This response, known as "SOS response", are induced by regulatory protein called RecA, which is activated when binds to single-strand DNA. DNA-RecA complex promotes the self-degradation of LexA, a common repressor for SOS genes.
+Microbes including ''Escherichia coli'' are known to respond to various DNA-injuring stress (ionizing radiation, ultraviolet radiation, peroxides etc...), altering their gene expression patterns. This response, known as the "SOS response", is induced by a regulatory protein called RecA when it binds to single-strand DNA. The DNA-RecA complex promotes the degradation of LexA, a common repressor of SOS genes.
-SulA is responsible for stress-induced halt of cell division. The promoter of sulA, or sulAp, is acutely induced by various stress factors, including ultraviolet irradiation.
+SulA is responsible for stress-induced halt of cell division. The promoter of ''sulA'', sulAp, is induced by various stress factors, including ultraviolet irradiation.
-We provide a [[BBa_K518013 |sulAp]] evaluation device to make it easy to measure the varying expression of [[BBa_K518013 |sulAp]] in various "stressful" conditions. Employing our [[Part:BBa_K518002 |dual luciferase assay kit]], quantitative measurement and comparison of Relative Promoter Unit (RPU) is achieved.
+We provide a [[Part:BBa_K518010 |sulAp]] evaluation device to make it easy to measure the varying expression of [[Part:BBa_K518010 |sulAp]] in various "stressful" conditions. Employing our [[Part:BBa_K518002 |dual luciferase assay kit]], quantitative measurement and comparison of Relative Promoter Unit (RPU) can be achieved.
-===Measurement===
+===Measurement1: Characterization of the [[Part:BBa_K518002 |dual luciferase assay kit (BBa_K518002)]] ===
+==Calibration for Firefly Luciferase==
+First, we carried out a calibration for ''Photynus pyralis'' (Firefly) luciferase using standard reagents of enzyme and D-luciferin. Reagents were granted from Berthold Japan (Tokyo, Japan). Luminescence was measured using a luminometer GENE LIGHT 200 from MICROTEC, Co., Ltd. (Chiba, Japan).
+The result indicates that under 1000 luciferase molecules can be detected, and that luciferase amounts can be well estimated by a simple linear regression in a logarithmically seven-digit range (from 10^(-20) to 10^(-13) mol). Again, we can easily get the numerical information of how many molecules exist, not just the arbitrary luminescence units.
+Since we can estimate the number of cells in a test tube from various data including OD600 value, we are able to estimate how many luciferase molecules are translated in a single bacterium on average.
+[[Image:Calibration1.png|500px]]
+<Fig 1. Estimation of firefly luciferase amount by linear regression.>
+==Test for [[Part:BBa_K518002 |BBa_K518002]] ==
+We first performed a test for [[Part:BBa_K518002 |BBa_K518002]] using [[Part:BBa_J23119 |BBa_J23119]] and [[Part:BBa_R0011 |BBa_R0011]].
+We successfully verified the IPTG-dependence of [[Part:BBa_R0011 |BBa_R0011]], and at the same time, the potency of [[Part:BBa_K518002 |BBa_K518002]] as an evaluation tool.
+For experimental and analytical details, see [http://2011.igem.org/Team:UT-Tokyo our page].
+<div style="float:left">[[Image:Firefly1.png |450px]]</div>
+[[Image:Firefly2.png |450px]]
+<div style="clear:both"></div>
+<Fig.1: Time-dependent luminescence measurement of firefly luciferase. The first graph shows the raw data obtained by triplicate experiments. Data is presented as mean ± S.D.. The second graph is a model fitting according to the enzymatic kinetics theory.>
+<div style="float:left">[[Image:Renilla1.png |450px]]</div>
+[[Image:Renilla2.png |450px]]
+<div style="clear:both"></div>
+<Fig.2: Time-dependent luminescence measurement of renilla luciferase.>
+[[Image:Promoter_Analysis.png |550px]]
+<Fig.3: Demonstration of promoter evaluation. Relative Promoter Unit (RPU) is calculated as: (maximum of firefly luminescence) / (maximum of renilla luminescence). Data is obtained from triplicate experiments. Data is expressed as mean±S.D..>
+<div style="clear:both"></div>
+===Measurement2: [[Part:BBa_K518010 |sulAp]] evaluation===
+The expression levels of sulAp were evaluated before and after UV induction. We evaluated this using both a ''recA''(-) (JM109) and a ''recA''(+) (BL21) strain. RecA is known to be necessary for releasing sulAp from repression. We successfully demonstrated a significant alteration of expression in recA(+) strain only after UV irradiation. The expression level of [[Part:BBa_J23119 |BBa_J23119]], a constitutive E. coli promoter which is often used as a comparison, was simultaneously presented as a comparison. Data is expressed as mean ± S.D.. Data is obtained from the average of three independent experiments.
 [[Image:sulApexpression.png]]
-<Figure: UV-induced expression levels of [[Part:BBa_K518010 |sulAp]]. The expression levels of sulAp were evaluated before and after UV induction. We evaluated it using both recA(-) (JM109) and recA(+) (BL21) strain. RecA is known as necessary for releasing sulAp from repression. We successfully demonstrate a significant alteration of expression in recA(+) strain after UV irradiation. The expression level of [[Part:BBa_J23119 |BBa_J23119]], a consensus sequence of E. coli promoters, was simultaneously presented as a comparison. Data is expressed as mean ± S.D.. Data is obtained from three independent experiments.>
+<Figure: UV-induced expression levels of [[Part:BBa_K518010 |sulAp]].>
 For experimental details, see [http://2011.igem.org/Team:UT-Tokyo our page].
-For detailed information on the dual luciferase assay kit, see [[Part:BBa_K518002 |BBa_K518002]] page.
 <!-- -->