Difference between revisions of "Team:TU Delft/Composite Part"
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<h2 class="title-style-1">Composite Parts<span class="title-under"></span></h2> | <h2 class="title-style-1">Composite Parts<span class="title-under"></span></h2> | ||
<div class="container col-md-10 col-md-offset-1"> | <div class="container col-md-10 col-md-offset-1"> | ||
| − | + | <h2 class="title-style-2">Producing microlenses with bacteria</h2> | |
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| − | + | <p>The essencial activity that our <i>Escherichia coli</i> needs to perfom to create biolenses surround | |
| − | + | itself by a glass layer. This is done by a special enzyme, silicatein-α, which is original from sponges and produces polysilicate | |
| − | < | + | from monomeric silicic acid. To make sure that the cell is coated by polysilicate we engineered a fusion protein combining |
| − | + | the silicatein-α gene from <i>Tethya aurantia</i> to the membrane protein OmpA (Outer membrane protein A) from <i>E. coli</i> | |
| − | + | (Part <strong><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1890002" target="_blank">K1890002</a></strong>) | |
| − | + | </p> | |
| − | + | <p>We expressed this construct under the control of an inducible promoter (Lac-promoter), which was present in the plasmid backbone | |
| − | + | we used, together with the LacI gene. This backbone was obtained from pBbA5c-RFP, a gift from Jay Keasling (Addgene plasmid # 35281) | |
| − | + | (<a href="#references">Lee <i>et al.</i>, 2011</a>). Upon transformation of this plasmid in BL21 <i>E. coli</i> cells | |
| − | + | and after induction with IPTG and supplementing the growth medium with silicic acid (the substrate for silicatein to produce | |
| − | + | the polysilicate layer), our cells were covered by a polysilicate layer as shown by Rhodamine 123 staining (a specific stain for | |
| − | + | polysilicate (Figure 1) and <a href="https://2016.igem.org/Team:TU_Delft/Project#silicatein" target="_blank"><b>other imaging experiments</b></a>. | |
| − | + | </p> | |
| − | + | <div class = "row"> | |
| − | + | <div class="col-md-10 col-md-offset-1 col-sm-12"> | |
| − | < | + | <figure> |
| − | + | <img src="https://static.igem.org/mediawiki/2016/8/8c/T--TU_Delft--silicatein92.png" alt="Rhodamine staining"> | |
| − | + | <figcaption><b>Figure 1:</b> Widefield and fluorescence images of OmpA-silicatein with silicic acid and OmpA-silicatein | |
| − | + | without silicic acid (negative control) at maximum excitation energy. Of the widefield and fluorescence images an | |
| − | + | overlay was made to show the fraction of fluorescent cells. The negative control causes overexposure of the camera, | |
| − | + | therefore the fluorescent image only gives one uniform signal. </figcaption> | |
| − | + | </figure> | |
| − | + | </div></div> | |
| − | + | <p>n figures 1 we can see that the strain transformed with OmpA-silicatein clearly has a different output from the negative | |
| − | + | control. The fluorescence of this sample is only localized at the cells. This might mean that the Rhodamine 123 has | |
| − | + | stained these cells and therefore the OmpA-silicatein cells could have the polysilicate layer around their membranes. </p> | |
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Revision as of 00:25, 20 October 2016
Composite Parts
Producing microlenses with bacteria
The essencial activity that our Escherichia coli needs to perfom to create biolenses surround itself by a glass layer. This is done by a special enzyme, silicatein-α, which is original from sponges and produces polysilicate from monomeric silicic acid. To make sure that the cell is coated by polysilicate we engineered a fusion protein combining the silicatein-α gene from Tethya aurantia to the membrane protein OmpA (Outer membrane protein A) from E. coli (Part K1890002)
We expressed this construct under the control of an inducible promoter (Lac-promoter), which was present in the plasmid backbone we used, together with the LacI gene. This backbone was obtained from pBbA5c-RFP, a gift from Jay Keasling (Addgene plasmid # 35281) (Lee et al., 2011). Upon transformation of this plasmid in BL21 E. coli cells and after induction with IPTG and supplementing the growth medium with silicic acid (the substrate for silicatein to produce the polysilicate layer), our cells were covered by a polysilicate layer as shown by Rhodamine 123 staining (a specific stain for polysilicate (Figure 1) and other imaging experiments.
n figures 1 we can see that the strain transformed with OmpA-silicatein clearly has a different output from the negative control. The fluorescence of this sample is only localized at the cells. This might mean that the Rhodamine 123 has stained these cells and therefore the OmpA-silicatein cells could have the polysilicate layer around their membranes.
