Difference between revisions of "Part:BBa K091100:Experience"

Latest revision as of 14:26, 21 September 2011

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UNIQa3aa0ad448341571-partinfo-00000000-QINU

•••••

[http://2011.igem.org/Team:KULeuven K.U.Leuven iGEM 2011 Team]

Characterization by K.U.Leuven 2011 iGEM Team

To gain insights into the usefulness of the lactose-inducible promoter I13453 in our 2011 iGEM project, we fused the promoter to a GFP reporter, and assayed the promoter’s activity after addition of different amounts of IPTG. IPTG is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon. Unlike lactose, IPTG cannot be metabolized by wild-type E.coli cells, leading to a constitutive high presence of the inducer. For this reason, IPTG is often used instead of lactose to induce the lac operon.

We tested the activity in a TOP10F’ (figure 1) as well as a MG1655 (figure 2) E.coli strain background. For more information on E.coli strain descriptions, we recommend the following [http://openwetware.org/wiki/E._coli_genotypes website].

Figure1

Addition of IPTG to TOP10F’ cells results in a minor growth defect, as can be seen in Figure 1A. However, this does not seem to inhibit the induction of the promoter, as Figure 1B clearly demonstrates that IPTG results in a clear induction of fluorescence, while without IPTG no such induction is seen.

Figure2

In MG1655 E.coli cells, IPTG at a concentration of 1.5mM leads to a minor growth defect (Figure 2A). Looking at the fluorescence measurements (Figure 2B), it is clear that the promoter already displays high activity in the absence of inducer, and that this activity is not increased by adding IPTG. Adding IPTG at concentrations of 1mM or 1.5mM results in an even lower fluorescence, and hence promoter activity, than the situation without IPTG addition. We think that this is due to the fact that we express our construct on a multicopy vector, which may outcompete the inhibitory activity of the LacI repressor under these conditions. In contrast to the MG1655 cells, the TOP10F’ strain contains a high copy LacI repressor, making the investigated promoter activity not leaky. These experiments highlight the importance of checking the strain background for compatibility with a desired system. The investigated promoter, pLac-Lux (I13453), should also be repressed by the luxR repressor bound to the corepressor CO6HSL. Due to time limitations, we could not investigate this aspect of the promoter.

As an additional control, we checked the activity of a constitutive promoter under the same conditions as described here. For results on these experiments, check out our BBa_K584001 page.

UNIQa3aa0ad448341571-partinfo-00000002-QINU

@@ Line 24: / Line 24: @@
 |width='60%' valign='top'|
 <u><b>Characterization by K.U.Leuven 2011 iGEM Team</b></u>
-<p align="justify">To gain insights into the usefulness of the lactose-inducible promoter I13453 in our 2011 iGEM project, we fused the promoter to a GFP reporter, and assayed the promoter’s activity after addition of different amounts of IPTG. IPTG is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon. Unlike lactose, IPTG cannot be metabolized by wild-type E.coli cells, leading to a constitutive high presence of inductor. For this reason, IPTG is often used instead of lactose to induce the lac operon.</p>
+<p align="justify">To gain insights into the usefulness of the lactose-inducible promoter I13453 in our 2011 iGEM project, we fused the promoter to a GFP reporter, and assayed the promoter’s activity after addition of different amounts of IPTG. IPTG is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon. Unlike lactose, IPTG cannot be metabolized by wild-type E.coli cells, leading to a constitutive high presence of the inducer. For this reason, IPTG is often used instead of lactose to induce the lac operon.</p>
-<p align="justify">We tested the activity both in a TOP10F’ (figure 1) as well as a MG1655 (figure 2) E.coli strain background. For more information on E.coli strain descriptions, we recommend the following  [http://openwetware.org/wiki/E._coli_genotypes website].</p>
+<p align="justify">We tested the activity in a TOP10F’ (figure 1) as well as a MG1655 (figure 2) E.coli strain background. For more information on E.coli strain descriptions, we recommend the following  [http://openwetware.org/wiki/E._coli_genotypes website].</p>
 [[Image:PLac TOP10F.jpg|thumb|center|Figure1]]
@@ Line 34: / Line 34: @@
 [[Image:pLac MG1655.jpg |height="500" width="500"|thumb|center|Figure2]]
-<p align="justify">In MG1655 E.coli cells, IPTG at a concentration of 1.5mM leads to a minor growth defect (Figure 2A). Looking at the fluorescence measurements (Figure 2B), it is clear that the promoter already displays high activity in the absence of inducer, and that this activity is not increased by adding IPTG. More still, adding IPTG at concentrations of 1mM or 1.5mM even results in a lower fluorescence, and hence promoter activity, than the situation without IPTG addition. We think that this is due to the fact that we express our construct on a multicopy vector, which may outcompete the inhibitory activity of the LacI repressor under these conditions. In contrast to the MG1655 cells, the TOP10F’ strain contains a high copy LacI repressor, making the investigated promoter activity not leaky. These experiments highlight the importance of checking the strain background for compatibility with a desired system.
+<p align="justify">In MG1655 E.coli cells, IPTG at a concentration of 1.5mM leads to a minor growth defect (Figure 2A). Looking at the fluorescence measurements (Figure 2B), it is clear that the promoter already displays high activity in the absence of inducer, and that this activity is not increased by adding IPTG. Adding IPTG at concentrations of 1mM or 1.5mM results in an even lower fluorescence, and hence promoter activity, than the situation without IPTG addition. We think that this is due to the fact that we express our construct on a multicopy vector, which may outcompete the inhibitory activity of the LacI repressor under these conditions. In contrast to the MG1655 cells, the TOP10F’ strain contains a high copy LacI repressor, making the investigated promoter activity not leaky. These experiments highlight the importance of checking the strain background for compatibility with a desired system.
 The investigated promoter, pLac-Lux (I13453), should also be repressed by the luxR repressor bound to the corepressor CO6HSL. Due to time limitations, we could not investigate this aspect of the promoter.</p>