Difference between revisions of "Part:BBa K864400"

Revision as of 19:52, 25 October 2016

Ptac, trp & lac regulated promoter

The Ptac promoter is a functional hybrid promoter, derived from the trp and lac promoters, that are regulated by trp and lac [1]. This part also exist together with lacI, part BBa_K180000

[1] Proc. Natl. Acad. Sci. USA, Vol. 80, pp. 21-25

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

INSA Lyon 2016 Experiments on this part

1. pTAC_RFP characterization

Description

pTAC promoter was taken from part BBa_K864400. This promoter is a hybrid of two operons: the trp and lac operons. This promoter is inducible by IPTG and commonly used in Escherichia coli for overproduction of proteins. E. coli NM522 strain that we used in our lab constitutively produces the LacIq protein, a strong pTAC promoter repressor. However, in absence of IPTG, we observed a strong leakage when plating our BBa_K1934000 transformants. Therefore, we decided to put a RFP reporter ORF under control of the pTAC promoter to characterize the promoter-driven transcriptional noise.

Expression of the pTAC-RFP fusion in presence of increasing amount of the inductor IPTG

Experimental design

The RFP coding sequence (BBa_E1010) was placed in silico under the control of the pTAC promoter (BBa_K864400), a strong RBS (BBa_B0030) and a bidirectional terminator (BBa_B0011). IDT performed the DNA synthesis and delivered the part as gBlock. The construct was cloned by conventional ligation into pSB1C3 plasmid and then transformed into E. coli NM522 strain. In order to study the efficiency of the pTAC promoter for the overproduction of proteins, recombinant clones were grown overnight in LB at 37°C in duplicate in three different induction conditions (IPTG concentrations): 0 mmol.L^-1, 1 mmol.L^-1 and 5 mmol.L^-1. OD₆₀₀ of each culture was measured every hour over six hours. E. coli NM522 strain was grown overnight in LB at 37°C in the same three induction conditions as control.

Results

Noise of the pTAC: fluorescence in absence of IPTG

We studied the noise of the promoter by comparing the normalized fluorescence between the construction and the NM522 strain without any induction of IPTG.

**Figure 1. Fluorescence/OD₆₀₀ comparison between NM522 with or without the plasmid (RFP).** Fluorescence of each sample was measured every hour over six hours with ChemiDoc Imaging system and was normalized by dividing by the OD₆₀₀. After one hour, a significant expression difference was noticed; NM522 strain with plasmid revealed a higher protein expression.

An ANOVA was made to see if there was a time effect between the two populations. We obtained a p-value of 0.61, suggesting that time had no effect on pTAC-RFP expression in absence of IPTG (α<0.05). Given this result, we gathered data to analyze if there was a significant difference between the two strains. A Student test was performed with the variance not equal. The p-value of 5.44*10^-4 indicated that the strain carrying pTAC-RFP transcriptional fusion displayed a higher fluorescence than the control strain.

pTAC induction by increasing concentration of IPTG

Then we studied the induction of the promoter by comparing the normalized fluorescence of the construction under the induction of [IPTG] = 0 and 1 mmol.L^-1.

**Figure 2. Fluorescence/OD₆₀₀ comparison between NM522 strains with plasmid (RFP) and with IPTG induction of 0 or 1 mmol.L^-1.** Fluorescence of each sample was measured every hour over six hours with ChemiDoc Imaging system and was normalized by dividing by the OD₆₀₀. After one hour, a significant expression difference was noticed; IPTG induction enables a higher protein expression rate.

An ANOVA test was made to see if there was a time effect between the two populations. A p-value of 0.21 indicated that time had no effect on the fluorescence induction (α<0.05). Data was therefore gathered in order to compare the strain fluorescence with 1 mmol.L^-1 IPTG and no IPTG. We realized a Student test with the variance not equal and obtained a p-value of 8.57*10^-3, showing a difference of fluorescence due to the presence of IPTG in the medium.

Finally, we compared the expression of the pTAC-RFP transcriptional fusion in 2 concentrations of IPTG: 1 and 5 mmol.L^-1.

**Figure 3. Fluorescence/OD₆₀₀ comparison between NM522 strains with plasmid (RFP) and with IPTG induction of 1 or 5 mmol.L^-1** Fluorescence of each sample was measured every hour over six hours with ChemiDoc Imaging system and was normalized by dividing by the OD₆₀₀. Even after six hours of incubation, no significant expression difference was observed, protein expression rate was not correlated with the concentration of IPTG.

An ANOVA was made to see if there was a time effect between the two populations. A p-value of 0.06 indicated that time had no effect (α<0.05). From there, we could gather the data to analyze if there was a significant difference between the two concentrations of IPTG.

We realized a Student test with the variance not equal and obtained a p-value of 0.61, indicating that no significant difference of fluorescence was observed with the rise of IPTG concentration.

Conclusion

In the case of RFP, the pTAC promoter seemed to not enable a gene tune because statistics showed that there was a significant noise, even in the absence of IPTG.

2. pTAC_CFP characterization

Description

pTAC promoter was taken from part part BBa_K864400 . This promoter is a hybrid of two operons: the trp and lac operons.This promoter is inducible by IPTG and commonly used in Escherichia coli for overproduction of proteins. Escherichia coli NM522 strain that we used in our lab constitutively express the LacIq protein, a strong pTAC promoter repressor. However, in absence of IPTG, we observed a strong leakage when plating our BBa_K1934000 transformants. Therefore, we decided to put a CFP reporter ORF under control of the pTAC promoter to characterize the promoter-driven transcriptional noise.

Expression of the pTAC-CFP fusion in presence of increasing amount of the inductor IPTG

Experimental design

The CFP coding sequence (BBa_E2020) was placed in silico under the control of the pTAC promoter (BBa_K864400), a strong RBS (BBa_B0030) and a bidirectional terminator (BBa_B0011). IDT performed the DNA synthesis and delivered the part as gBlock. The construct was cloned by conventional ligation into pSB1C3 plasmid and then transformed into E. coli NM522 strain. In order to study the efficiency of the pTAC promoter for the overproduction of proteins, recombinant clones were grown overnight in LB at 37°C in duplicate in three different induction conditions (IPTG concentrations): 0 mmol.L^-1, 1 mmol.L^-1 and 5 mmol.L^-1. OD₆₀₀ of each culture was measured every hour over six hours. E. coli NM522 strain was grown overnight in LB at 37°C in the same three induction conditions as control.

Results

Noise of the pTAC: fluorescence in absence of IPTG

We studied the noise of the promoter by comparing the normalized fluorescence between the construction and the NM522 strain without any induction of IPTG.

**Figure 1. Fluorescence/OD₆₀₀ comparison between NM522 with or without the plasmid (CFP).** Fluorescence of each sample was measured every hour over six hours with ChemiDoc Imaging system and was normalized by dividing by the OD₆₀₀. After one hour, a significant expression difference was noticed; NM522 strain with plasmid revealed a higher protein expression.

An ANOVA was made to see if there was a time effect between the two populations. We obtain a p-value of 0.96, suggesting that time had no effect on pTAC-CFP expression in absence of IPTG (α<0.05). Given this result, we gathered data to analyze if there was a significant difference between the two strains. A Student test was performed with the variance not equal. The p-value of 0.10 indicated that the strain carrying pTAC-CFP transcriptional fusion didn’t display a significant fluorescence difference with the control strain.

pTAC induction by increasing concentration of IPTG

Then we studied the induction of the promoter by comparing the normalized fluorescence of the construction under the induction of [IPTG] = 0 and 1 mmol.L^-1.

**Figure 2. Fluorescence/OD₆₀₀ comparison between NM522 strains with plasmid (CFP) and with IPTG induction of 0 or 1 mmol.L^-1.** Fluorescence of each sample was measured every hour over six hours with ChemiDoc Imaging system and was normalized by dividing by the OD₆₀₀. After one hour, a significant expression difference was noticed; IPTG induction enabled a higher protein expression rate.

An ANOVA test was made to see if there was a time effect between the two populations. A p-value of 0.05 indicated that time had no effect on the fluorescence induction (α<0.05). Data was therefore gathered in order to compare the strain fluorescence with 1 mmol.L^-1 IPTG and no IPTG. We realized a Student test with the variance not equal and obtained a p-value of 4.64*10^-10, showing a significant difference of fluorescence due to the presence of IPTG in the medium.

Finally, we compared the expression of the pTAC-CFP transcriptional fusion in 2 concentrations of IPTG: 1 and 5 mmol.L^-1.

**Figure 3. Fluorescence/OD₆₀₀ comparison between NM522 strains with plasmid (CFP) and with IPTG induction of 1 or 5 mmol.L^-1** Fluorescence of each sample was measured every hour during six hours with ChemiDoc Imaging system and was normalized by dividing by the OD₆₀₀. Even after six hours of incubation, no significant expression difference could be detected, protein expression rate was not correlated with the concentration of IPTG.

An ANOVA was made to see if there was a time effect between the two populations. A p-value of 0.55 indicated that time had no effect (α<0.05). From there, we gathered data to analyze if there was a significant difference between the two concentrations of IPTG. We realized a Student test with the variance not equal and obtained a p-value of 0.54, indicating that no significant difference of fluorescence was observed with the rise of IPTG concentration.

Conclusion

In the case of CFP, the pTAC promoter seemed to enable a gene tune because there wasn’t a differential gene expression in absence of IPTG, so a significant noise wasn’t measured.

@@ Line 24: / Line 24: @@
 <html>
 <h3 id="CBD">Description</h3>
-<p>The pTAC promoter used corresponds to the part BBa_K864400. This promoter is a hybrid of two operons: the trp and lac operons.This promoter is  inducible by IPTG and  commonly used in <i>Escherichia coli</i> for overproduction of proteins. <i>Escherichia coli</i> NM522 strain that we used in our lab constitutively express the LacIq protein, a strong pTAC promoter repressor. However, in absence of IPTG, we observed  a strong leakage when plating our <a href="https://parts.igem.org/Part:BBa_K1934000">BBa_K1934000</a> transformants . Therefore, we decided to put a RFP reporter ORF under control of the pTAC promoter to characterize the promoter noise.
+<p>pTAC promoter was taken from part <a href="https://parts.igem.org/Part:BBa_K864400">BBa_K864400</a>. This promoter is a hybrid of two operons: the trp and lac operons. This promoter is  inducible by IPTG and  commonly used in <i>Escherichia coli</i> for overproduction of proteins. <i>E. coli</i> NM522 strain that we used in our lab constitutively produces the LacIq protein, a strong pTAC promoter repressor. However, in absence of IPTG, we observed a strong leakage when plating our <a href="https://parts.igem.org/Part:BBa_K1934000">BBa_K1934000</a> transformants. Therefore, we decided to put a RFP reporter ORF under control of the pTAC promoter to characterize the promoter-driven transcriptional noise.
 </p>
@@ Line 31: / Line 31: @@
 <h5 id="CBD">Experimental design</h5>
-The RFP coding sequence <a href="https://parts.igem.org/Part:BBa_E1010">(BBa_E1010)</a> was placed in silico under the control of the pTAC promoter <a href="https://parts.igem.org/Part:BBa_K864400">(BBa_K864400)</a>, a strong RBS <a href="https://parts.igem.org/Part:BBa_B0030">(BBa_B0030)</a> and a bidirectional terminator <a href="https://parts.igem.org/Part:BBa_B0011">(BBa_B0011)</a>. IDT realized the DNA synthesis and delivered the part as gBlock. The construct was cloned by conventional ligation into pSB1C3 iGEM reference plasmid and then transformed into <i>Escherichia coli</i> NM522 strain.
+The RFP coding sequence (<a href="https://parts.igem.org/Part:BBa_E1010">BBa_E1010</a>) was placed <i>in silico</i> under the control of the pTAC promoter (<a href="https://parts.igem.org/Part:BBa_K864400">BBa_K864400</a>), a strong RBS (<a href="https://parts.igem.org/Part:BBa_B0030">BBa_B0030</a>) and a bidirectional terminator (<a href="https://parts.igem.org/Part:BBa_B0011">BBa_B0011</a>). IDT performed the DNA synthesis and delivered the part as gBlock. The construct was cloned by conventional ligation into pSB1C3 plasmid and then transformed into <i>E. coli</i> NM522 strain.
-In order to study the efficiency of pTAC promoter for proteins overproduction, recombinant clones were grown overnight in LB at 37°C in duplicate in three different induction conditions: 0 mmol.L<sup>-1</sup>, 1 mmol.L<sup>-1</sup> and 5 mmol.L<sup>-1</sup>. The OD<sub>600</sub> of each culture was measured each hour during six hours.
+In order to study the efficiency of the pTAC promoter for the overproduction of proteins, recombinant clones were grown overnight in LB at 37°C in duplicate in three different induction conditions (IPTG concentrations): 0 mmol.L<sup>-1</sup>, 1 mmol.L<sup>-1</sup> and 5 mmol.L<sup>-1</sup>. OD<sub>600</sub> of each culture was measured every hour over six hours.
-<i>Escherichia coli</i> NM522 strain was grown overnight in LB at 37°C in the same three induction conditions as control.
+<i>E. coli</i> NM522 strain was grown overnight in LB at 37°C in the same three induction conditions as control.
@@ Line 44: / Line 44: @@
 </ul>
-<p>We study the noise of the promoter by comparing the normalized fluorescence between the construction and the NM522 strain without any induction of IPTG.</p>
+<p>We studied the noise of the promoter by comparing the normalized fluorescence between the construction and the NM522 strain without any induction of IPTG.</p>
-<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/2016/f/fc/INSA-Lyon_RFP-NM522.jpg" width = "800"/><figcaption><b>Figure 1. Fluorescence/DO600 comparison between NM522 with or without the plasmid (RFP). </b> Fluorescence of each sample was measured every hour during six hours with ChemiDoc image system and was normalized by dividing by the OD600. After one hour, a significant expression difference is remarkable; NM522 strain with plasmid reveals a higher protein expression. </figcaption></figure>
+<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/2016/f/fc/INSA-Lyon_RFP-NM522.jpg" width = "800"/><figcaption><b>Figure 1. Fluorescence/OD<sub>600</sub> comparison between NM522 with or without the plasmid (RFP). </b> Fluorescence of each sample was measured every hour over six hours with ChemiDoc Imaging system and was normalized by dividing by the OD<sub>600</sub>. After one hour, a significant expression difference was noticed; NM522 strain with plasmid revealed a higher protein expression. </figcaption></figure>
-<p>An ANOVA was made to see if there was a time effect between the two populations. We obtain a p-value of 0.61, suggesting that the time have no effect on pTAC-RFP expression in absence of IPTG (α<0.05). Given this result, we can gather the data to analyse if there is a significant difference between the two strains.
+<p>An ANOVA was made to see if there was a time effect between the two populations. We obtained a p-value of 0.61, suggesting that time had no effect on pTAC-RFP expression in absence of IPTG (α<0.05). Given this result, we gathered data to analyze if there was a significant difference between the two strains.
-A Student test was performed with the variance not equal. The p-value of 5.44*10<sup>-4</sup> indicates that the strain carrying pTAC-RFP transcriptional fusion display a higher fluorescence than the control strain.</p>
+A Student test was performed with the variance not equal. The p-value of 5.44*10<sup>-4</sup> indicated that the strain carrying pTAC-RFP transcriptional fusion displayed a higher fluorescence than the control strain.</p>
 <ul style="list-style-type:circle">
@@ Line 55: / Line 55: @@
 </ul>
-<p>We now study the induction of the promoter by comparing the normalized fluorescence of the construction under the induction of [IPTG] = 0 and 1 mmol.L<sup>-1</sup>.</p>
+<p>Then we studied the induction of the promoter by comparing the normalized fluorescence of the construction under the induction of [IPTG] = 0 and 1 mmol.L<sup>-1</sup>.</p>
-<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/2016/b/bd/INSA-Lyon_0vs1.jpg" width = "800"/><figcaption><b>Figure 2. Fluorescence/DO600 comparison between NM522 strains with plasmid (RFP) and with IPTG induction of 0 or 1 mmol.L<sup>-1</sup>. </b> Fluorescence of each sample was measured every hour during six hours with ChemiDoc image system and was normalized by dividing by the OD600. After one hour, a significant expression difference is remarkable; IPTG induction enables a higher protein expression rate. </figcaption></figure>
+<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/2016/b/bd/INSA-Lyon_0vs1.jpg" width = "800"/><figcaption><b>Figure 2. Fluorescence/OD<sub>600</sub> comparison between NM522 strains with plasmid (RFP) and with IPTG induction of 0 or 1 mmol.L<sup>-1</sup>. </b> Fluorescence of each sample was measured every hour over six hours with ChemiDoc Imaging system and was normalized by dividing by the OD<sub>600</sub>. After one hour, a significant expression difference was noticed; IPTG induction enables a higher protein expression rate. </figcaption></figure>
-<p>An ANOVA test was made to see if there was a time effect between the two populations. The p-value of 0.21 indicates that the time have no effect on the fluorescence induction (α<0.05). The data were therefore gathered in order to compare the strain fluorescence with 1 mmol.L<sup>-1</sup> IPTG and without.
+<p>An ANOVA test was made to see if there was a time effect between the two populations. A p-value of 0.21 indicated that time had no effect on the fluorescence induction (α<0.05). Data was therefore gathered in order to compare the strain fluorescence with 1 mmol.L<sup>-1</sup> IPTG and no IPTG.
-We realize a Student test with the variance not equal and obtain a p-value of 8.57*10<sup>-3</sup>, showing a difference of fluorescence due to the presence of IPTG in the medium.</p>
+We realized a Student test with the variance not equal and obtained a p-value of 8.57*10<sup>-3</sup>, showing a difference of fluorescence due to the presence of IPTG in the medium.</p>
 <p>Finally, we compared the expression of the pTAC-RFP transcriptional fusion in 2 concentrations of IPTG: 1 and 5 mmol.L<sup>-1</sup>.</p>
-<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/2016/6/64/INSA-Lyon_1vs5.jpg" width = "800"/><figcaption><b>Figure 3. Fluorescence/DO600 comparison between NM522 strains with plasmid (RFP) and with IPTG induction of 1 or 5 mmol.L<sup>-1</sup> </b> Fluorescence of each sample was measured every hour during six hours with ChemiDoc image system and was normalized by dividing by the OD600. Even after six hours of incubation, a significant expression difference can't be detected, the protein expression rate is not correlated with the concentration of IPTG. </figcaption></figure>
+<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/2016/6/64/INSA-Lyon_1vs5.jpg" width = "800"/><figcaption><b>Figure 3. Fluorescence/OD<sub>600</sub> comparison between NM522 strains with plasmid (RFP) and with IPTG induction of 1 or 5 mmol.L<sup>-1</sup> </b> Fluorescence of each sample was measured every hour over six hours with ChemiDoc Imaging system and was normalized by dividing by the OD<sub>600</sub>. Even after six hours of incubation, no significant expression difference was observed, protein expression rate was not correlated with the concentration of IPTG. </figcaption></figure>
-<p>An ANOVA was made to see if there was a time effect between the two populations. The p-value of 0.06 indicates that the time have no effect (α<0.05). From there, we can gather the data to analyse if there is a significant difference between the two concentrations of IPTG.</p>
+<p>An ANOVA was made to see if there was a time effect between the two populations. A p-value of 0.06 indicated that time had no effect (α<0.05). From there, we could gather the data to analyze if there was a significant difference between the two concentrations of IPTG.</p>
-<p>We realize a Student test with the variance not equal and obtain a p-value of 0.61, indicating that no significant difference of fluorescence was observed with the rise of IPTG concentration.</p>
+<p>We realized a Student test with the variance not equal and obtained a p-value of 0.61, indicating that no significant difference of fluorescence was observed with the rise of IPTG concentration.</p>
 <h3 id="CBD">Conclusion</h3>
-<p>In the case of RFP, the pTAC promoter seems to not enable a gene tune because statistics show that there is a significant noise, even in absence of IPTG.</p>
+<p>In the case of RFP, the pTAC promoter seemed to not enable a gene tune because statistics showed that there was a significant noise, even in the absence of IPTG.</p>
 <h2> <strong>2. <a href"https://parts.igem.org/Part:BBa_K1934050">pTAC_CFP</a> characterization </strong></h2>
-<html>
 <h3 id="CBD">Description</h3>
-<p>The pTAC promoter used corresponds to the part BBa_K864400. This promoter is a hybrid of two operons: the trp and lac operons.This promoter is  inducible by IPTG and  commonly used in <i>Escherichia coli</i> for overproduction of proteins. <i>Escherichia coli</i> NM522 strain that we used in our lab constitutively express the LacIq protein, a strong pTAC promoter repressor. However, in absence of IPTG, we observed  a strong leakage when plating our <a href="https://parts.igem.org/Part:BBa_K1934000">BBa_K1934000</a> transformants. Therefore, we decided to put a CFP reporter ORF under control of the pTAC promoter to characterize the promoter noise.</p>
+<h3 id="CBD">Description</h3>
+<p>pTAC promoter was taken from part <a href="https://parts.igem.org/Part:BBa_K864400">part BBa_K864400</a> . This promoter is a hybrid of two operons: the trp and lac operons.This promoter is  inducible by IPTG and  commonly used in <i>Escherichia coli</i> for overproduction of proteins. <i>Escherichia coli</i> NM522 strain that we used in our lab constitutively express the LacIq protein, a strong pTAC promoter repressor. However, in absence of IPTG, we observed  a strong leakage when plating our <a href="https://parts.igem.org/Part:BBa_K1934000">BBa_K1934000</a> transformants. Therefore, we decided to put a CFP reporter ORF under control of the pTAC promoter to characterize the promoter-driven transcriptional noise.
 <h3 id="CBD">Expression of the pTAC-CFP fusion in presence of increasing amount of the inductor IPTG</h3>
@@ Line 83: / Line 82: @@
 <h5 id="CBD">Experimental design</h5>
-The CFP coding sequence <a href="https://parts.igem.org/Part:BBa_E2020">(BBa_E2020)</a> was placed in silico under the control of the pTAC promoter <a href="https://parts.igem.org/Part:BBa_K864400">(BBa_K864400)</a>, a strong RBS <a href="https://parts.igem.org/Part:BBa_B0030">(BBa_B0030)</a> and a bidirectional terminator <a href="https://parts.igem.org/Part:BBa_B0011">(BBa_B0011)</a>. IDT realized the DNA synthesis and delivered the part as gBlock. The construct was cloned by conventional ligation into pSB1C3 iGEM reference plasmid and then transformed into <i>Escherichia coli</i> NM522 strain.
+The CFP coding sequence (<a href="https://parts.igem.org/Part:BBa_E2020">BBa_E2020</a>) was placed <i>in silico</i> under the control of the pTAC promoter (<a href="https://parts.igem.org/Part:BBa_K864400">BBa_K864400</a>), a strong RBS (<a href="https://parts.igem.org/Part:BBa_B0030">BBa_B0030</a>) and a bidirectional terminator (<a href="https://parts.igem.org/Part:BBa_B0011">BBa_B0011</a>). IDT performed the DNA synthesis and delivered the part as gBlock. The construct was cloned by conventional ligation into pSB1C3 plasmid and then transformed into <i>E. coli</i> NM522 strain.
-In order to study the efficiency of pTAC promoter for proteins overproduction, recombinant clones were grown overnight in LB at 37°C in duplicate in three different induction conditions: 0 mmol.L<sup>-1</sup>, 1 mmol.L<sup>-1</sup> and 5 mmol.L<sup>-1</sup>. The OD<sub>600</sub> of each culture was measured each hour during six hours.
+In order to study the efficiency of the pTAC promoter for the overproduction of proteins, recombinant clones were grown overnight in LB at 37°C in duplicate in three different induction conditions (IPTG concentrations): 0 mmol.L<sup>-1</sup>, 1 mmol.L<sup>-1</sup> and 5 mmol.L<sup>-1</sup>. OD<sub>600</sub> of each culture was measured every hour over six hours.
-<i>Escherichia coli</i> NM522 strain was grown overnight in LB at 37°C in the same three induction conditions as control.
+<i>E. coli</i> NM522 strain was grown overnight in LB at 37°C in the same three induction conditions as control.
@@ Line 96: / Line 95: @@
 </ul>
-<p>We study the noise of the promoter by comparing the normalized fluorescence between the construction and the NM522 strain without any induction of IPTG.</p>
+<p>We studied the noise of the promoter by comparing the normalized fluorescence between the construction and the NM522 strain without any induction of IPTG.</p>
-<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/5/59/T--INSA-Lyon--CFPvsNM522.jpeg" width = "800"/><figcaption><b>Figure 4. Fluorescence/DO600 comparison between NM522 with or without the plasmid (CFP). </b> Fluorescence of each sample was measured every hour during six hours with ChemiDoc image system and was normalized by dividing by the OD600. After one hour, a significant expression difference is remarkable; NM522 strain with plasmid reveals a higher protein expression. </figcaption></figure>
+<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/5/59/T--INSA-Lyon--CFPvsNM522.jpeg" width = "800"/><figcaption><b>Figure 1. Fluorescence/OD<sub>600</sub> comparison between NM522 with or without the plasmid (CFP). </b> Fluorescence of each sample was measured every hour over six hours with ChemiDoc Imaging system and was normalized by dividing by the OD<sub>600</sub>. After one hour, a significant expression difference was noticed; NM522 strain with plasmid revealed a higher protein expression. </figcaption></figure>
-<p>An ANOVA was made to see if there was a time effect between the two populations. We obtain a p-value of 0.96, suggesting that the time have no effect on pTAC-CFP expression in absence of IPTG (α<0.05). Given this result, we can gather the data to analyse if there is a significant difference between the two strains.
-A Student test was performed with the variance not equal. The p-value of 0.10 indicates that the strain carrying pTAC-CFP transcriptional fusion doesn’t display a significant fluorescence difference with the control strain.</p>
+<p>An ANOVA was made to see if there was a time effect between the two populations. We obtain a p-value of 0.96, suggesting that time had no effect on pTAC-CFP expression in absence of IPTG (α<0.05). Given this result, we gathered data to analyze if there was a significant difference between the two strains.
+A Student test was performed with the variance not equal. The p-value of 0.10 indicated that the strain carrying pTAC-CFP transcriptional fusion didn’t display a significant fluorescence difference with the control strain.</p>
 <ul style="list-style-type:circle">
@@ Line 107: / Line 107: @@
 </ul>
-<p>We now study the induction of the promoter by comparing the normalized fluorescence of the construction under the induction of [IPTG] = 0 and 1 mmol.L<sup>-1</sup>.</p>
+<p>Then we studied the induction of the promoter by comparing the normalized fluorescence of the construction under the induction of [IPTG] = 0 and 1 mmol.L<sup>-1</sup>.</p>
-<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/0/02/T--INSA-Lyon--CFP_0vs1.jpeg" width = "800"/><figcaption><b>Figure 5. Fluorescence/DO600 comparison between NM522 strains with plasmid (CFP) and with IPTG induction of 0 or 1 mmol.L<sup>-1</sup>. </b> Fluorescence of each sample was measured every hour during six hours with ChemiDoc image system and was normalized by dividing by the OD600. After one hour, a significant expression difference is remarkable; IPTG induction enables a higher protein expression rate. </figcaption></figure>
+<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/0/02/T--INSA-Lyon--CFP_0vs1.jpeg" width = "800"/><figcaption><b>Figure 2. Fluorescence/OD<sub>600</sub> comparison between NM522 strains with plasmid (CFP) and with IPTG induction of 0 or 1 mmol.L<sup>-1</sup>. </b> Fluorescence of each sample was measured every hour over six hours with ChemiDoc Imaging system and was normalized by dividing by the OD<sub>600</sub>. After one hour, a significant expression difference was noticed; IPTG induction enabled a higher protein expression rate. </figcaption></figure>
-<p>An ANOVA test was made to see if there was a time effect between the two populations. The p-value of 0.05 indicates that the time have no effect on the fluorescence induction (α<0.05). The data were therefore gathered in order to compare the strain fluorescence with 1 mmol.L<sup>-1</sup> IPTG and without.
+<p>An ANOVA test was made to see if there was a time effect between the two populations. A p-value of 0.05 indicated that time had no effect on the fluorescence induction (α<0.05). Data was therefore gathered in order to compare the strain fluorescence with 1 mmol.L<sup>-1</sup> IPTG and no IPTG.
-We realize a Student test with the variance not equal and obtain a p-value of 4.64*10<sup>-10</sup>, showing a significant difference of fluorescence due to the presence of IPTG in the medium.</p>
+We realized a Student test with the variance not equal and obtained a p-value of 4.64*10<sup>-10</sup>, showing a significant difference of fluorescence due to the presence of IPTG in the medium.</p>
 <p>Finally, we compared the expression of the pTAC-CFP transcriptional fusion in 2 concentrations of IPTG: 1 and 5 mmol.L<sup>-1</sup>.</p>
-<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/a/a4/T--INSA-Lyon--CFP_1vs5.jpeg" width = "800"/><figcaption><b>Figure 6. Fluorescence/DO600 comparison between NM522 strains with plasmid (CFP) and with IPTG induction of 1 or 5 mmol.L<sup>-1</sup> </b> Fluorescence of each sample was measured every hour during six hours with ChemiDoc image system and was normalized by dividing by the OD600. Even after six hours of incubation, a significant expression difference can't be detected, the protein expression rate is not correlated with the concentration of IPTG. </figcaption></figure>
+<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/a/a4/T--INSA-Lyon--CFP_1vs5.jpeg" width = "800"/><figcaption><b>Figure 3. Fluorescence/OD<sub>600</sub> comparison between NM522 strains with plasmid (CFP) and with IPTG induction of 1 or 5 mmol.L<sup>-1</sup> </b> Fluorescence of each sample was measured every hour during six hours with ChemiDoc Imaging system and was normalized by dividing by the OD<sub>600</sub>. Even after six hours of incubation, no significant expression difference could be detected, protein expression rate was not correlated with the concentration of IPTG. </figcaption></figure>
-<p>An ANOVA was made to see if there was a time effect between the two populations. The p-value of 0.55 indicates that the time have no effect (α<0.05). From there, we can gather the data to analyse if there is a significant difference between the two concentrations of IPTG.
+<p>An ANOVA was made to see if there was a time effect between the two populations. A p-value of 0.55 indicated that time had no effect (α<0.05). From there, we gathered data to analyze if there was a significant difference between the two concentrations of IPTG.
-We realize a Student test with the variance not equal and obtain a p-value of 0.54, indicating that no significant difference of fluorescence was observed with the rise of IPTG concentration.
+We realized a Student test with the variance not equal and obtained a p-value of 0.54, indicating that no significant difference of fluorescence was observed with the rise of IPTG concentration.
 </p>
 <h3 id="CBD">Conclusion</h3>
-<p>In the case of CFP, the pTAC promoter seems to enable a gene tune because there isn’t a differential gene expression in absence of IPTG, so a significant noise isn’t measured.</p>
+<p>In the case of CFP, the pTAC promoter seemed to enable a gene tune because there wasn’t a differential gene expression in absence of IPTG, so a significant noise wasn’t measured.</p>
-<h2> <strong>3. Protein nature influence on promoter behavior </strong></h2>
-After studying promoter noise and difference of protein expression with variable induction conditions ([IPTG] = 0, 1 and 5 mmol.L[sup]-1[/sup]), it was interested to compare the promoter behavior according to the protein nature (CFP or RFP).
-<h5 id="CBD">Results</h5>
-<ul style="list-style-type:circle">
-  <li><b>Noise of the pTAC: fluorescence in absence of IPTG</b></li>
-</ul>
-<p>We study the noise of the promoter by comparing the normalized fluorescence between NM522 strain with RFP or CFP construction without any induction of IPTG.</p>
-<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/2/20/T--INSA_Lyon--Comparaison_0IPTG.png" width = "800"/><figcaption><b>Figure 7. Fluorescence/DO600 comparison between NM522 with the plasmids RFP or CFP without induction. </b> Fluorescence of each sample was measured each hour during six hours with ChemiDoc image system and was normalized by dividing by the OD600. After six hours, a significant expression difference isn't remarkable; NM522 strains with both plasmids reveal a similar protein expression. </figcaption></figure>
-<p>An ANOVA was made to see if there was a time effect between the two populations. We obtain a p-value of 0.89, suggesting that the time have no effect on pTAC-RFP and pTAC-CFP expression in absence of IPTG (α<0.05). Given this result, we can gather the data to analyse if there is a significant difference between the two strains.
-A Student test was performed with the variance not equal. The p-value of 0.38 indicates that there isn't any significant difference of expression rate between strains carrying pTAC-RFP or pTAC-CFP transcriptional fusions.</p>
-<ul style="list-style-type:circle">
-  <li><b>Differential protein expression rate with pTAC induction by IPTG</b></li>
-</ul>
-<p>We now study the differential protein expression rate with IPTG induction of the promoter by comparing the normalized fluorescence of the two constructions under the induction of [IPTG] = 1 mmol.L<sup>-1</sup>.</p>
-<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/b/bf/T--INSA_Lyon--Comparaison_1IPTG.png" width = "800"/><figcaption><b>Figure 8. Fluorescence/DO600 comparison between NM522 with the plasmids RFP or CFP with [IPTG] = 1 mmol.L-1. </b> Fluorescence of each sample was measured each hour during six hours with ChemiDoc image system and was normalized by dividing by the OD600. After six hours, a significant expression difference isn't remarkable; NM522 strains with both plasmids reveal a similar protein expression. </figcaption></figure>
-<p>An ANOVA was made to see if there was a time effect between the two populations. We obtain a p-value of 0.70, suggesting that the time have no effect on pTAC-RFP and pTAC-CFP expression in presence of IPTG (α<0.05). Given this result, we can gather the data to analyse if there is a significant difference between the two strains.</p>
-<p>A Student test was performed with the variance not equal. The p-value of 0.84 indicates that there isn't any significant difference of expression rate between strains carrying pTAC-RFP or pTAC-CFP transcriptional fusions.</p>
-<h3 id="CBD">Conclusion</h3>
-<p>In the cases of RFP and CFP, the pTAC promoter seems to enable a similar gene expression in absence and in presence of IPTG.</p>
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