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| − | <partinfo>BBa_K1486002 short</partinfo> | + | <partinfo>MCf_C1486032 short</partinfo> |
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| − | https://static.igem.org/mediawiki/2014/0/06/Sfgfpcpxr_gradient.jpg
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| − | ===Purpose of the Biobrick=== | + | ===Purpose of the Unit=== |
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| | <FONT SIZE="2"><P> | | <FONT SIZE="2"><P> |
| − | This construct aimed to evaluate the expression of our CpxR construct, and the function of the arabinose promoter in E coli by fusing a superfolder GFP protein to the N terminus of CpxR. The sfGFP was chosen because of its higher intensity compared to GFP. | + | This unit (in the control layer) is a button used to apply pressure on the cells in the chamber. It was designed to decrease the outflow of cells into the channels. |
| − | Not knowing if CpxR would react the same way if sfGFP were attached to the N or C terminus, 2 biobricks were built, one with each of the orientations.
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| − | <FONT SIZE="2"><P>
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| − | An experiment on both possible CpxR - sfGFP orientations was launched to determine whether the proteins were well expressed and folded, and if the arabinose promoter worked well. The experiment conditions can be found here. The basics were the following: increasing concentrations of arabinose as shown in Table 1 and results of a plate reader with excitation and emission wavelengths of 490 nm and 510 nm respectively.
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| − | https://static.igem.org/mediawiki/2014/1/10/Screen_Shot_2014-10-13_at_16.20.13_.png
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| − | </P></FONT>
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| − | https://static.igem.org/mediawiki/2014/9/98/Sfgfpcpxr_N_plot.png
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| − | <FONT SIZE="2"><P>
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| − | The results of this experiment show that CpxR is well expressed, as GFP can be seen. Also, the results show that the arabinose promoter works as expected.
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| − | </P></FONT>
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| − | <FONT SIZE="3"><P>
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| − | ===Microfluidics===
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| − | </P></FONT>
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| − | <FONT SIZE="2"><P>A characterization experiment in a microfluidic chip was also done. The aim of this experiment was to induce CpxR expression with arabinose.</P></FONT>
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| − | <FONT SIZE="2"><P>Cells were inoculated and grown overnight in 3 ml LB medium with chloramphenicol. The next morning the cells were loaded on chip and the upper half of the chip had a flow of LB with arabinose and chloramphenicol whereas the lower half of the chip had a flow of LB with only chloramphenicol. The detailed protocol can be found on our <a href="http://2014.igem.org/Team:EPF_Lausanne/Notebook/Microfluidics">wiki's microfluidics page.</a></P></FONT>
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| − | https://static.igem.org/mediawiki/2014/0/0d/Truc1.png
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| − | <FONT SIZE="1"><P>This is a schematic representation of how the bacteria were divided on the chip. M1 and M2 stands for multiplex 1 and 2 and are valves that allow to block the flow of bacteria (or medium). Thanks to these multiplexes up to 16 different types of cells/bacteria can be flown in distinct rows. In this experiment we only used a separation in two region (ara+ and ara-). </P></FONT>
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| − | https://static.igem.org/mediawiki/2014/4/4e/Truc2.png
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| − | <FONT SIZE="1"><P>Figure 1. Scan of the microfluidic chip at t = 0min. As expected, no signal is detected. </P></FONT>
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| − | https://static.igem.org/mediawiki/2014/3/32/Truc3.png
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| − | <FONT SIZE="1"><P>Figure 2. Scan of the microfluidic chip at t = 300min. The upper half of the chip has medium with arabinose and the lower half doesn't. Expression is detected on the upper half. </P></FONT>
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| − | <FONT SIZE="2"><P>After GenePix analysis, where we calculated the intensity of each chamber and also the intensity of the area next to the chamber (to subtract as background value) we calculated an average of fluorescence expression. Figure 3 shows the results. </P></FONT>
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| − | https://static.igem.org/mediawiki/2014/4/4c/Gfp_ara.png
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| − | <FONT SIZE="1"><P>Figure 3. Evolution of CpxR-GFP fluorescence over time</P></FONT>
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| − | <FONT SIZE="2"><P>A great GFP signal was detected which confirms the expression of our portein of interest sfGFP-Cpxr.</P></FONT>
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| − | <FONT SIZE="2"><P>The increasing standard deviation for the cells with arabinose can be explained as some chambers did not have a lot of cells and so there was a low intensity. As it can be seen in the following picture :</P></FONT>
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| − | https://static.igem.org/mediawiki/2014/f/f7/Truc5.png
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| − | <FONT SIZE="1"><P>Figure 4. These are chambers with arabinose in the medium, you can see that there are different cell density and thus different intensity in the chambers. Inducing a high standard deviation</P></FONT>
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| − | <!-- Add more about the biology of this part here
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| − | ===Usage and Biology===
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| − | <span class='h3bb'>Sequence and Features</span>
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| − | <partinfo>BBa_K1486002 SequenceAndFeatures</partinfo>
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| − | <!-- Uncomment this to enable Functional Parameter display
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| | ===Functional Parameters=== | | ===Functional Parameters=== |
| − | <partinfo>BBa_K1486002 parameters</partinfo>
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