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| - | mKate2 is a red fluorescent protein that we want to use as a acceptor for FRET. According to Evrogen <sup><span style="color:blue">[2]</span></sup>
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| - | mKate2 has an excitation maximum at 588 nm and an emission maximum at 633 nm. The
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| - | diagram below (graph 2, colored dots) shows that the excitation maximum is at 579 nm and the
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| - | emission maximum is 611 nm. The first peak does not look exactly like a single peak, but we can see a
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| - | clear maximum. The problem in our FRET system is the very low intensity which results
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| - | in the next diagram (graph 3) in a huge difference of intensity of the two fluorescence
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| - | proteins.<br><br>
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| - | </p>
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| - | <center><img alt="Spectra mKate" src="/wiki/images/f/fa/Spectra_mKate.png" width="800" height="400"></center> <br>
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| - | <center>Graph 2: "BL21DE3pLys[pBR-IBA2-mKate]":<br>
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| - | Excitation spectrum (dashed line) and emission spectrum (solid line) of
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| - | mKate with marked maximums<br><br></center><br><br>
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| - | <br><br>
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| - | <center><img alt="mKate bac" src="/wiki/images/3/3f/Mkatepicbact.png" width="400" height="500"></center> <br>
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| - | <center>Graph 2.1: "BL21DE3pLys[pBR-IBA2-mKate]":<br>
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| - | Pellet of E.coli Bl21(DE3) cells with expressed mKate. Left side: without UV radiation, right side: with UV radiation.<br><br></center><br><br>
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| - | <br><br>
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| - | <b>FRET:</b><br>
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| - | <p text-aligne:left style="margin-left:50px; margin-right:50px">
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| - | Both diagrams in an overlay shows us the FRET system. LSSmOrange is the donor and mKate is the
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| - | acceptor. For an optimum in FRET, it should display a stronger overlap between the emission maximum of LSSmOrange and the excitation maximum of mKate.<br><br>
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| - | </p>
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| - | <center><img alt="Spectra FRET" src="/wiki/images/4/41/FRET_Normalized_2.png" width="800" height="400"></center> <br>
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| - | <center>Graph 3: "FRET pair LSSmOrange-mKate":<br>
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| - | Normalized excitation and emission spectra overlay of of graph 1 and graph 2.<br><br></center><br><br>
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| - | <br>
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| - | <p text-aligne:left style="margin-left:50px; margin-right:50px">
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| - | The next figure is from the paper of M. Shcherbakova. In this figure one can
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| - | clearly see the overlap between donor and acceptor in an optimal FRET system. <br><br>
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| - |
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| - | </p>
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| - | <center><img alt="Spectra" src="/wiki/images/6/6c/ShcherbakovaSpectra.png" width="400" height="349"></center> <br>
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| - | <center>Excitation and emission spectra overlay of donor LSSmOrange and acceptor mKate2 <sup><span style="color:blue">[1]</span></sup><br>
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| - | (dashed line = excitation spectrum, solid line = emission spectrum)</center><br><br>
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| - | <br><br><br>
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| - | <font size="3" color="#F0F8FF" face="Arial regular">
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| - | <p text-aligne:left style="margin-left:50px; margin-right:50px">
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| - |
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| - | <center><b>Growth curves:</b></center>
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| - | <br>
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| - | <p text-aligne:left style="margin-left:50px; margin-right:50px">
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| - | To determine the influence of the induced constructs mKate and LSSmOrange on the BL21DE3, growth curves were used. During a time period of 8 h the OD of the samples was measured at a wavelength of 600 nm in time steps of 15 min. The measurement was started at an OD<sub>600 nm</sub> value of 0.3. With the help of the following formulas the growth rate k and the doubling time t<sub>d</sub> were calculated:
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| - | <br><br>
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| - | </p>
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| - | <center>
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| - | <img alt="Spectra" src="/wiki/images/5/52/Johanna_Formel.JPG" width="200" height="100">
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| - | </center>
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| - | <br>
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| - |
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| - | <font size="3" color="#F0F8FF" face="Arial regular">
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| - | <p text-aligne:left style="margin-left:50px; margin-right:50px">
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| - |
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| - | The calculated values of k and td of the competent cells BL21DE3 containing the mKate or LSSmOrange construct were compared to the level of the wild type strain, to reveal possible toxicity of the constructs. In the following diagrams the OD<sub>600</sub>, respectively the ln OD<sub>600</sub>, values are plotted against the time and in the table below the resulting values of the growth rates k and the doubling times td are listed. <br><br>
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| - |
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| - | <center><img alt="Growth curve" src="/wiki/images/6/65/WACHSTUMSKURVE2.jpg" width="800" height="400"></center> <br>
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| - | <center>Graph 4: "Growth curves":<br>
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| - | <center>Growth curves of BL21DE3 WT, BL21DE3 mKate and BL21DE3 LSSmOrange. The OD<sub>600</sub> values are plotted against the time [min]. </center><br><br>
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| - |
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| - |
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| - | <br>
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| - | <center><img alt="Growth curve2" src="/wiki/images/a/a1/WACHSTUMSKURVE2-LOG.jpg" width="800" height="400"></center> <br>
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| - | <center>Graph 5: "Growth curves log":<br>
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| - | <center>Semi-logarithmic growth curves of BL21DE3 WT, BL21DE3 mKate and BL21DE3 LSSmOrange. The ln (OD<sub>600</sub> values) are plotted against the time [min].</center><br>
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