Team:Hong Kong HKUST/pneumosensor/characterization

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(Difference between revisions)

Latest revision as of 22:28, 17 October 2014

Pneumosensor Characterization

σ^x(BBa_K1379004)

Introduction

To test the functionality of σ^X, we first enable constitutive expression of σ^X in the σ^X Generator, BBa_K1379006. The generator was then assembled with the standard promoter measurement kit BBa_E0240, with either promoter P_celA ( Promoter only: BBa_K1379000, w/ BBa_E0240: BBa_K1379002) and P_comFA (Promoter only: BBa_K1379001, w/ BBa_E0240: BBa_K1379003). E. coli colonies holding the resulting constructs in pSB3K3 were observed under fluorescent macroscope with UV filter. Measurement kit for standard reference promoter BBa_J23101, which is BBa_I20260 was used as a positive control; BBa_E0240 was used as the general negative control for background fluorescence. Measurement kits for P_celA and P_comFA without σ^X Generator were used as negative controls for function of σ^X.

Results

Figure 1. P_celA and P_comFA promoters activated in presence of σ^X.

Only in the presence of σ^X would P_celA and P_comFA be turned on, as GFP expression could be seen when σ^X is present. Therefore, σ^X is functional. P_celA and P_comFA gave little GFP signal in the absence of σ^X but has comparable activity as reference promoter BBa_J23101 in presence of σ^X. Scale bar = 5mm.

P_celA (BBa_K1379000) and P_comFA (BBa_K1379001)

For characterization, P_celA promoter was assembled with the promoter measurement kit BBa_E0240 to give the P_celA Measurement Kit BBa_K1379002 in plasmid pSB3K3. The construct was further assembled with σ^X generator BBa_K1379006 to give BBa_K1379005.

P_comFA promoter was assembled with the promoter measurement kit BBa_E0240 to give the P_comFA Measurement Kit BBa_K1379003 in plasmid pSB3K3. The construct was further assembled with σ^X generator BBa_K1379006 to give BBa_K1379007.

Qualitative characterization was performed by comparing intensities of GFP signals from colonies of E. coli DH10B strain holding the P_celA and P_comFA Measurement Kits with and without the σ^X generator under a fluorescent macroscope with UV filter. Measurement kit for standard reference promoter BBa_J23101, BBa_I20260 was used as a positive control; BBa_E0240 was used as the negative control for background fluorescence.

Quantitative characterization was performed following the protocol described in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). E. coli DH10B strains holding the constructs with or without σ^X generator respectively were grown to mid-log phases. GFP intensities and cell densities were then sampled every 30 minutes for 5 consecutive time points to obtain growth rates and GFP synthesis rates. The GFP synthesis rates were then compared to that of standard reference promoter BBa_J23101 measurement device BBa_I20260 to obtain the Relative Promoter Units (RPUs). For subtraction of background fluorescence, pSB3K3 holding BBa_E0240 was measured alongside. The measurement was done with 3 replicas.

Figure 2. P_celA has 0.53 RPU and P_comFA hsa 1.21 RPU when paired with σ^X generator.

P_celA and P_comFA was measured in reference to BBa_J23101 constitutive promoter with and without σ^X generator BBa_K1379006. RPU shown was calculated from 3 replicas.

Characterization Method

Construction

1. Construct pSB3K3-σ^x Generator-(BBa_K1379006)-P_celA-BBa_E0240
Or migrate BBa_K1379005 to pSB3k3.

2. Construct pSB3K3-σ^x Generator-(BBa_K1379006)-P_comFA-BBa_E0240
Or migrate BBa_K1379007 to pSB3K3.

3. Transforming pSB3K3-P_celA-BBa_E0240

4. Transforming pSB3K3-P_comFA-BBa_E0240

5. Transforming pSB3K3-BBa_I20260 (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit

6. Transforming pSB3K3BBa_E0240 (GFP generator) from the 2014 Distribution Kit.

Measurement

1. Preparing supplemented M9 medium
(M9 Minimal salt medium protocols could be seen on the Protocols page, or download the PDF file)

2. Culturing E. coli DH10B strain carrying the whole construct listed on the construction part. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)

3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium in the 96 Deep Well plate.

4. Incubate in 37°C and shake for 3 - 4 hours.

5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)

6. Measuring the GFP intensity and OD595 values (we used Envision Multilabel Reader) every 30 minutes after the above mentioned E. coli strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)

Filter used on Envision Multilabel Reader:
- Absorbance :Photometric 595nm,
- Excitation :485nm FITC,
- Emission :535nm FITC,
- Mirror module : FITC (403) at bottom.

- In between measurements, keep incubating the cells in 37°C while shaking.

7. Calculating the Relative Promoter Units (RPU) using the obtained data;

Data Processing

1. After E. coli carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min);

2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-BBa_E0240. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken;

3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve;

4. Absolute promoter activity of P_celA, P_comFA, and BBa_I20260 were calculated by dividing the GFP synthesis rate with the average OD600 value;

5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values;

6. Finally, R.P.U was calculated by dividing the averaged P_celA and P_comFA absolute promoter activity over the averaged BBa_J23101 absolute promoter activity. R.P.U value of P_celA and P_comFA reflect the maximum GFP expression in the presence of σ^x. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P_celA and P_comFA promoter in the absence of σ^x.

Equation of the RPU calculation is shown below:

References

BioCyc was retrieved from http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867 and http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867

Luo P., & Morrison D. (2003). Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae. Journal of Bacteriology. doi:10.1128/JB.185.1.349-358.2003

Piotrowski A., Luo P., & Morrison D. (2009). Competence for genetic transformation in Streptococcus pneumoniae: termination of activity of the alternative sigma factor ComX is independent of proteolysis of ComX and ComW. Journal of Bacteriology. doi:10.1128/JB.01750-08

Rhodius V., Segall-Shapiro T., Sharon B., Ghodasara A., Orlova E., Tabakh H., . . . Voigt C. (2013). Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters. Molecular Systhetic Biology .doi:10.1038/msb.2013.58

J. R. Kelly, A. J. Rubin, J. H. Davis, J. Cumbers, M. J. Czar, ..., D. Endy. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. Journal of Biological Engineering, 3, 4. doi: 10.1186/1754-1611-3-4

Team:Hong Kong HKUST/pneumosensor/characterization

From 2014.igem.org

Latest revision as of 22:28, 17 October 2014

Pneumosensor Characterization

σ^x(BBa_K1379004)

Figure 1. P_celA and P_comFA promoters activated in presence of σ^X.

P_celA (BBa_K1379000) and P_comFA (BBa_K1379001)

Figure 2. P_celA has 0.53 RPU and P_comFA hsa 1.21 RPU when paired with σ^X generator.

P_celA and P_comFA was measured in reference to BBa_J23101 constitutive promoter with and without σ^X generator BBa_K1379006. RPU shown was calculated from 3 replicas.

Characterization Method

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 		<h2>Pneumosensor Characterization</h2>
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 			<div class= "content_area_two_row">
-				<p><br><b>Introduction</b> <br><br>
+				<p><br><b><u>Introduction</u></b> <br><br>
-<p class="first_letter_enhanced">To test the functionality of &sigma;<sup>X</sup>, we first enable constitutive expression of &sigma;<sup>X</sup> in the &sigma;<sup>X</sup> Generator, BBa_K1379006. The generator was then assembled with the standard promoter measurement kit BBa_E0240, with either promoter P<sub>chbB</sub> (Promoter only: BBa_K1379000, w/ BBa_E0240: BBa_K1379002) and P<sub>comFA</sub> (Promoter only: BBa_K1379001, w/ BBa_E0240: BBa_K1379003). <i>E. coli</i> colonies holding the resulting constructs in pSB3K3 were observed under fluorescent macroscope with UV filter. Measurement kit for standard reference promoter BBa_J23101,  which is I20260 was used as a positive control; BBa_E0240 was used as the general negative control. for background fluorescence. Measurement kits for P<sub>chbB</sub> P<sub>comFA</sub> without &sigma;<sup>X</sup> Generator were used as negative controls for function of &sigma;<sup>X</sup>.
+<p class="first_letter_enhanced">To test the functionality of &sigma;<sup>X</sup>, we first enable constitutive expression of &sigma;<sup>X</sup> in the &sigma;<sup>X</sup> Generator, <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>.
+The generator was then assembled with the standard promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>, with either promoter P<sub>celA</sub> (
+Promoter only: <a href= "http://parts.igem.org/Part:BBa_K1379000">BBa_K1379000</a>, w/ <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>:
+<a href= "http://parts.igem.org/Part:BBa_K1379002">BBa_K1379002</a>) and P<sub>comFA</sub> (Promoter only: <a href= "http://parts.igem.org/Part:BBa_K1379001">BBa_K1379001</a>,
+w/ <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>: <a href= "http://parts.igem.org/Part:BBa_K1379003">BBa_K1379003</a>). <i>E. coli</i> colonies holding the resulting
+ constructs in pSB3K3 were observed under fluorescent macroscope with UV filter. Measurement kit for standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">
+ BBa_J23101</a>,  which is <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> was used as a positive control; <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240
+ </a> was used as the general negative control for background fluorescence. Measurement kits for P<sub>celA</sub> and P<sub>comFA</sub> without &sigma;<sup>X</sup> Generator were used
+ as negative controls for function of &sigma;<sup>X</sup>.
 <br>
-</p><br><br>
+</p>
-<p><br><b>Results</b> <br><br></p>
+<p><br><b><u>Results</u></b> <br><br></p>
 <div class="content_image">
-						<img src= "https://static.igem.org/mediawiki/2014/4/43/PxhvV%26PcomFA_WIKI_copy.png"/>
+						<img src= "https://static.igem.org/mediawiki/2014/7/7f/PcelA%26comFA_macroscope.png" >
-							<h5 style="font-size: 13px">Figure 1. P<sub>chbB</sub> and P<sub>comFA</sub> promoters activated in presence of &sigma;<sup>X</sup>.</h5>
+							<h5 style="font-size: 13px">Figure 1. P<sub>celA</sub> and P<sub>comFA</sub> promoters activated in presence of &sigma;<sup>X</sup>.</h5>
-						<h6 style= "font-size: 13px"> Only in the presence of &sigma;<sup>X</sup> would P<sub>chbB</sub> and P<sub>comFA</sub> be turned on, therefore, &sigma;<sup>X</sup> is functional.  P<sub>chbB</sub> and P<sub>comFA</sub> gave little GFP signal in the absence of &sigma;<sup>X</sup> but has comparable activity as reference promoter BBa_J23101 in presence of &sigma;<sup>X</sup>. Scale bar = 5mm.</h6>
+						<h6 style= "font-size: 13px"> Only in the presence of &sigma;<sup>X</sup> would P<sub>celA</sub> and P<sub>comFA</sub> be turned on, as GFP expression could
+						be seen when &sigma;<sup>X</sup> is present. Therefore, &sigma;<sup>X</sup> is functional.  P<sub>celA</sub> and P<sub>comFA</sub> gave little GFP signal in the
+						absence of &sigma;<sup>X</sup> but has comparable activity as reference promoter BBa_J23101 in presence of &sigma;<sup>X</sup>. Scale bar = 5mm.</h6>
 						</div>
 </div>
@@ Line 48: / Line 54: @@
 		<!-- one row of content , two column one picture right-->
-		<div class='content_1'><h3> <a href= "http://parts.igem.org/Part:BBa_K1379000">P<sub>chbB</sub> (BBa_K1379000) </a></h3>
+		<div class='content_1'><h3> <a href= "http://parts.igem.org/Part:BBa_K1379000">P<sub>celA</sub> (BBa_K1379000)</a> and <a href= "http://parts.igem.org/Part:BBa_K1379001">P<sub>comFA</sub> (BBa_K1379001) </a></h3>
 		<table class="content_table" align= "center" >
-		<tr class= "content_row">
-			<td class= "content_cell">
+		<tr class= "content_row" valign= "top">
-				<div class= "content_area_one_row">
+			<td class= "content_cell" colspan= "2">
-					<p class="first_letter_enhanced">To measure the R.P.U (Relative Promoter Unit) of P<sub>chbB</sub> promoter, we adopted the method described by Kelly et al. in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). By linking P<sub>chbB</sub> promoter with GFP generator (BBa_E0240), the promoter activity was represented by the GFP synthesis rate. Fluorescence was measured using EnVision multilabel reader from Perkin Elmer Company, when the cells are in the mid-log phase. OD595 values were measured and converted to OD600 to obtain the R.P.U of the promoter. The σ<sup>x</sup> gene and P<sub>chbB</sub> promoter used in the construct are both cloned from <i>E. coli</i> NCTC 7465 strain. The experimental construct used was BBa_K1379005, which contain σ<sup>x</sup> generator, P<sub>chbB</sub>, and GFP generator. The positive control used in this characterization is BBa_I20260 which is a constitutive promoter containing GFP generator, while the negative control used in this characterization is BBa_K1379002 which is P<sub>chbB</sub> promoter with GFP generator but without σ<sup>x</sup> generator.<br>  </p>
+			<div class= "content_area_two_row">
+					<p class="first_letter_enhanced"> <br><br>For characterization, P<sub>celA</sub> promoter was assembled with the promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to give the P<sub>celA</sub> Measurement Kit <a href= "http://parts.igem.org/Part:BBa_K1379002">BBa_K1379002</a> in plasmid pSB3K3. The construct was further assembled with &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a> to give <a href= "http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a>. <br><br>
+P<sub>comFA</sub> promoter was assembled with the promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to give the P<sub>comFA</sub> Measurement Kit <a href= "http://parts.igem.org/Part:BBa_K1379003">BBa_K1379003</a> in plasmid pSB3K3. The construct was further assembled with &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a> to give <a href= "http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a>. <br><br>
-				</div>
+Qualitative characterization was performed by comparing intensities of GFP signals from colonies of <i>E. coli</i> DH10B strain holding the P<sub>celA</sub> and P<sub>comFA</sub> Measurement Kits with and without the &sigma;<sup>X</sup> generator under a fluorescent macroscope with UV filter. Measurement kit for standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> was used as a positive control; <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was used as the negative control for background fluorescence. <br><br>
-			</td>
-			<td class= "content_cell">
+Quantitative characterization was performed following the protocol described in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). <i>E. coli</i> DH10B strains holding the constructs with or without &sigma;<sup>X</sup> generator respectively were grown to mid-log phases. GFP intensities and cell densities were then sampled every 30 minutes for 5 consecutive time points to obtain growth rates and GFP synthesis rates. The GFP synthesis rates were then compared to that of standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> measurement device <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> to obtain the Relative Promoter Units (RPUs). For subtraction of background fluorescence, pSB3K3 holding <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was measured alongside. The measurement was done with 3 replicas.
+				</div><br><br>
 				<div class= "content_area_one_row">
 					<div class="content_image">
-						<img src= "https://static.igem.org/mediawiki/2014/6/6d/PchbB_prism_Large.png"/><br>
+						<img style="width:80%; display: block;
-						<h5 style="font-size: 13px">Figure 2. P<sub>chbB</sub> promoter Relative Promoter Unit (RPU) is measured with reference to J23101 constitutive promoter.</h5>
+    margin-left: auto;
-						<h6 style= "font-size: 13px"> P<sub>chbB</sub> promoter induced by σ<sup>x</sup> gave GFP signals. This fluorescence expression was measured over time, and divided by the OD of the cells. In the end, it was standardized based on J23101 promoter strength. Measurement was done by using 3 replicas.</h6>
+    margin-right: auto" src= "https://static.igem.org/mediawiki/2014/8/8c/Wiki_hkust_pcelA_pcomFA.png"/><br>
+						<h5 style="font-size: 13px">Figure 2. P<sub>celA</sub> has 0.53 RPU and P<sub>comFA</sub> hsa 1.21 RPU when paired with &sigma;<sup>X</sup> generator.</h5>
+						<h6 style= "font-size: 13px"> P<sub>celA</sub> and P<sub>comFA</sub> was measured in reference to <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> constitutive promoter with and without &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>. RPU shown was calculated from 3 replicas.</h6> <br><br>
 					</div>
@@ Line 76: / Line 89: @@
 		</div>
 <!-- one row of content , two column one picture right-->
-		<div class='content_1'><h3>P<sub>chbB</sub> Characterization Method</a></h3>
+		<div class='content_1'><h3>Characterization Method</h3>
 		<table class="content_table" align= "center" >
 		<tr class= "content_row">
 			<td class= "content_cell">
-				<div class= "content_area_one_row">
+			<div class= "content_area_one_row">
-<p><br><u><b>Characterization Procedure</b></u><br><br>
-. Constructing BBa_K1379005-pSB3K3 (σ<sup>x</sup> Generator (BBa_K1379006)-P<sub>chbB</sub>-BBa_E0240-pSB3K3) ,Transforming BBa_K1379002-pSB3K3 (P<sub>chbB</sub>-BBa_E0240-pSB3K3), Transforming BBa_I20260-pSB3K3 (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit, Transforming BBa_E0240-pSB3K3 (GFP generator) from the 2014 Distribution Kit.<br><br>
-. Preparing supplemented M9 medium (M9 Minimal salt medium protocols could be seen on the protocols page) <br><br>
+<p><u><b>Construction</b></u><br><br>
+. Construct pSB3K3-σ<sup>x</sup> Generator-(<a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>)-P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br>
-. Culturing <i>E. coli</i> DH10B strain carrying BBa_K1379005-pSB3K3, BBa_K1379002-pSB3K3, BBa_I20260-pSB3K3, BBa_E0240-pSB3K3 in supplemented M9 medium and measuring the respective growth curve; <br><br>
+Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a> to pSB3k3.<br><br>
-. Measuring the GFP intensity and OD595 values every 30 minutes after the above mentioned E. coli strains are cultured to mid-log phase; <br><br>
+. Construct pSB3K3-σ<sup>x</sup> Generator-(<a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>)-P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> <br>
-. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br>
+Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a> to pSB3K3.<br><br>
-</p>
-<p><br><u><b>Data Processing</b></u><br><br>
+. Transforming pSB3K3-P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br>
-. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br>
-. For GFP intensity, GFP expression of BBa_K1379005-pSB3K3, BBa_K1379002-PSB3K3, and BBa_I20260-pSB3K3 are subtracted with the background fluorescence which is BBa_E0240-pSB3K3. Curve reflecting GFP expression change was plotted; OD595 was converted to OD600, and average values were taken; <br><br>
+. Transforming pSB3K3-P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br>
-. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br>
+. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit<br><br>
-. Absolute promoter activity of P<sub>chbB</sub> and I20260 were calculated by dividing the GFP synthesis rate of BBa_K1379005-pSB3K3, BBa_K1379002-PSB3K3, and BBa_I20260-pSB3K3 over the average OD600 value; <br><br>
+. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit.<br><br>
+<br>
-. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br>
+<p><u><b>Measurement</b></u><br><br>
+. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br>
-. Finally, R.P.U was calculated by dividing the averaged P<sub>chbB</sub> absolute promoter activity over the averaged I20260 absolute promoter activity. R.P.U value of BBa_K1379005-pSB3K3 reflect the maximum GFP expression of PchbB promoter in the presence of σ<sup>x</sup>. Leakage could be analyzed according to the R.P.U value of BBa_K1379002-PSB3K3 which shows the GFP expression of P<sub>chbB</sub> promoter without σ<sup>x</sup>.<br><br>
+. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on the construction part. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br>
-</p>
+. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium in the 96 Deep Well plate. <br><br>
-				</div>
-			</td>
-			</tr>
-			</table>
+. Incubate in 37°C and shake for 3 - 4 hours.<br><br>
+. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br>
-		<!-- one row of content , two column one picture right-->
+. Measuring the GFP intensity and OD595 values (we used Envision Multilabel Reader) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br>
-		<!-- one row of content , two column one picture left-->
-		<div class='content_1'><h3><a href= "http://parts.igem.org/Part:BBa_K1379001"> P<sub>comFA</sub>(BBa_K1379001) </a> </h3>
-		<table class="content_table" align= "center" >
-		<tr class= "content_row">
-			<td class= "content_cell">
-				<div class= "content_area_one_row">
-					<div class="content_image">
-						<img src= "https://static.igem.org/mediawiki/2014/f/fd/PcomFA_prism_large.png"/>
-						<h5 style="font-size: 13px">Figure 3. P<sub>comFA</sub> promoter Relative Promoter Unit (RPU) is measured with reference to J23101 constitutive promoter. </h5>
-						<h6 style= "font-size: 13px"> P<sub>helicase</sub> promoter induced by σ<sup>x</sup> gave GFP signals. This fluorescence expression was measured over time, and divided by the OD of the cells. In the end, it was standardized based on J23101 promoter strength. Measurement was done by using 3 replicas.</h6>
-					</div>
-				</div>
-			</td>
-			<td class= "content_cell">
-				<div class= "content_area_one_row">
-					<p class="first_letter_enhanced">The method of measuring RPU (Relative Promoter Unit) of P<sub>comFA</sub> promoter is similar to measuring RPU of P<sub>chbB</sub> which adopted the method described by Kelly et al. in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). There are no difference in measurement condition of measuring P<sub>comFA</sub> and P<sub>chbB</sub>. Fluorescence and absorbance are measured in the same time period. The experimental construct used was BBa_K1379007, which contain σ<sup>x</sup> generator, P<sub>comFA</sub>, and GFP generator. The positive control used in this characterization is BBa_I20260 which is a constitutive promoter containing GFP generator, while the negative control used in this characterization is BBa_K1379002 which is P<sub>comFA</sub> promoter with GFP generator but without σ<sup>x</sup> generator.  </p>
-				</div>
-			</td>
-		</tr>
+Filter used on Envision Multilabel Reader: <br>
-		</div>
+- Absorbance :Photometric 595nm,<br>
-		</table>
+- Excitation :485nm FITC,<br>
-		</div>
+- Emission :535nm FITC, <br>
+- Mirror module : FITC (403) at bottom. <br><br>
+- In between measurements, keep incubating the cells in 37°C while shaking. <br><br>
+. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br>
-<!-- one row of content , two column one picture right-->
+</p>
-		<div class='content_1'><h3> P<sub>comFA</sub> Characterization Method</a></h3>
-		<table class="content_table" align= "center" >
-		<tr class= "content_row">
-			<td class= "content_cell">
-				<div class= "content_area_one_row">
-<p><br><u><b>Characterization Procedure</b></u><br><br>
-. Constructing BBa_K1379007-pSB3K3(σ<sup>x</sup> Generator (BBa_K1379006)-P<sub>comFA</sub>-BBa_E0240-pSB3K3) ,Transforming BBa_K1379003-pSB3K3 (P<sub>comFA</sub>-BBa_E0240-pSB3K3), Transforming BBa_I20260-pSB3K3 (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit, Transforming BBa_E0240-pSB3K3 (GFP generator) from the 2014 Distribution Kit.<br><br>
-. Preparing supplemented M9 medium (M9 Minimal salt medium protocols could be seen on the protocols page) <br><br>
-. Culturing <i>E. coli</i> DH10B strain carrying BBa_K1379007-pSB3K3, BBa_K1379003-pSB3K3, BBa_I20260-pSB3K3, BBa_E0240-pSB3K3 in supplemented M9 medium and measuring the respective growth curve; <br><br>
-. Measuring the GFP intensity and OD595 values every 30 minutes after the above mentioned E. coli strains are cultured to mid-log phase; <br><br>
-. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br>
-</p>
 <p><br><u><b>Data Processing</b></u><br><br>
 . After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br>
-. For GFP intensity, GFP expression of BBa_K1379007-pSB3K3, BBa_K1379003-PSB3K3, and BBa_I20260-pSB3K3 are subtracted with the background fluorescence which is BBa_E0240-pSB3K3. Curve reflecting GFP expression change was plotted; OD595 was converted to OD600, and average values were taken; <br><br>
+. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br>
 . GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br>
-. Absolute promoter activity of P<sub>comFA</sub> and I20260 were calculated by dividing the GFP synthesis rate of BBa_K1379007-pSB3K3, BBa_K1379003-PSB3K3, and BBa_I20260-pSB3K3 over the average OD600 value; <br><br>
+. Absolute promoter activity of P<sub>celA</sub>, P<sub>comFA</sub>, and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br>
 . Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br>
-. Finally, R.P.U was calculated by dividing the averaged P<sub>comFA</sub> absolute promoter activity over the averaged I20260 absolute promoter activity. R.P.U value of BBa_K1379007-pSB3K3 reflect the maximum GFP expression of PcomFA promoter in the presence of σ<sup>x</sup>. Leakage could be analyzed according to the R.P.U value of BBa_K1379003-PSB3K3 which shows the GFP expression of P<sub>comFA</sub> promoter without σ<sup>x</sup>.<br><br>
+. Finally, R.P.U was calculated by dividing the averaged P<sub>celA</sub> and P<sub>comFA</sub> absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>celA</sub> and P<sub>comFA</sub> reflect the maximum GFP expression in the presence of σ<sup>x</sup>. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>celA</sub> and P<sub>comFA</sub> promoter in the absence of σ<sup>x</sup>.<br><br>
+Equation of the RPU calculation is shown below: <br>
+<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/>
+<br><br>
 </p>
 				</div>
-			</td>
+			</td></tr></table></div>
+<br><br>
@@ Line 200: / Line 179: @@
 						<p>
 <br>
-						<u>References</u></p>
+						<u><b>References</b></u><br><br></p>
-						<p>J. R. Kelly, A. J. Rubin, J. H. Davis, J. Cumbers, M. J. Czar, ..., D. Endy. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. <i>Journal of Biological Engineering</i>, 3, 4. doi: 10.1186/1754-1611-3-4</p>
+						<p>
+BioCyc was retrieved from http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867 and http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867
+<br><br>
+Luo P., & Morrison D. (2003).<i> Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae</i>. Journal of Bacteriology. doi:10.1128/JB.185.1.349-358.2003
+<br><br>
+Piotrowski A., Luo P., & Morrison D. (2009). <i>Competence for genetic transformation in Streptococcus pneumoniae: termination of activity of the alternative sigma factor ComX is independent of proteolysis of ComX and ComW.</i> Journal of Bacteriology. doi:10.1128/JB.01750-08
+<br><br>
+Rhodius V., Segall-Shapiro T., Sharon B., Ghodasara A., Orlova E., Tabakh H., . . . Voigt C. (2013). <i>Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters.</i> Molecular Systhetic Biology .doi:10.1038/msb.2013.58
+<br><br>
+J. R. Kelly, A. J. Rubin, J. H. Davis, J. Cumbers, M. J. Czar, ..., D. Endy. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. <i>Journal of Biological Engineering</i>, 3, 4. doi: 10.1186/1754-1611-3-4
+</p>
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Team:Hong Kong HKUST/pneumosensor/characterization

From 2014.igem.org

Latest revision as of 22:28, 17 October 2014

Pneumosensor Characterization

Figure 1. PcelA and PcomFA promoters activated in presence of σX.

PcelA (BBa_K1379000) and PcomFA (BBa_K1379001)

Figure 2. PcelA has 0.53 RPU and PcomFA hsa 1.21 RPU when paired with σX generator.

PcelA and PcomFA was measured in reference to BBa_J23101 constitutive promoter with and without σX generator BBa_K1379006. RPU shown was calculated from 3 replicas.

Characterization Method

Figure 1. P_celA and P_comFA promoters activated in presence of σ^X.

P_celA (BBa_K1379000) and P_comFA (BBa_K1379001)

Figure 2. P_celA has 0.53 RPU and P_comFA hsa 1.21 RPU when paired with σ^X generator.

P_celA and P_comFA was measured in reference to BBa_J23101 constitutive promoter with and without σ^X generator BBa_K1379006. RPU shown was calculated from 3 replicas.