Difference between revisions of "Part:BBa K1934050"

Latest revision as of 19:36, 25 October 2016

CFP under the control of pTAC promoter

This part is composed of the CFP coding sequence under the control of the pTAC promoter, a strong RBS and a bidirectional terminator.

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]

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 14: / Line 14: @@
 <html>
 <h3 id="CBD">Description</h3>
-<p>The pTAC promoter used corresponds to the <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 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 <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>
+<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 20: / Line 20: @@
 <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 33: / Line 33: @@
 </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 1. Fluorescence/DO600 comparison between NM522 with or without the plasmid (CFP). </b> Fluorescence of each sample was measured very 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.
+<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 indicates that the strain carrying pTAC-CFP transcriptional fusion doesn’t display a significant fluorescence difference with the control strain.</p>
+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 45: / Line 45: @@
 </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 2. 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 very 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 3. 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 very 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>
 </html>