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| | <partinfo>BBa_K584002 short</partinfo> | | <partinfo>BBa_K584002 short</partinfo> |
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| − | This part was designed to test the functionality of the promotor region.
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| | <u><b>Characterization</b></u> | | <u><b>Characterization</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>
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| − | <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>
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| − | [[Image:PLac TOP10F.jpg|thumb|center|Figure1]]
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| − | <p align="justify">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.</p>
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| − | [[Image:pLac MG1655.jpg |height="500" width="500"|thumb|center|Figure2]]
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| − | <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.
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| − | 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>
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| − | <p align="justify">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 [https://parts.igem.org/wiki/index.php?title=Part:BBa_K584001 BBa_K584001] page.</p>
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| | + | <p align="justify">The <b>[http://2011.igem.org/Team:KULeuven K.U.Leuven 2011 iGEM Team]</b> has <b><u>extensively characterized</u></b> the <b><u>existing Biobrick part</u></b> [https://parts.igem.org/Part:BBa_K091100 pLac_lux hybrid promoter(BBa_K091100)].</p> |
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| | + | The detailed qualitative and quantitative <u><b>[https://parts.igem.org/Part:BBa_K091100:Experience#User_Reviews review]</b></u> has been added into the experience page of the BBa_K091100 PBad promoter biobrick. |
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