Team:TU Eindhoven/Design/Membrane Anchor Design

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                   <h2>Microfluidics: Introduction</h2>
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                   <h2>Membrane Anchor Design</h2>
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                   <p>A substantial part of the TU Eindhoven iGEM 2014 Project is Microfluidics. Microfluidics is a technique that comprises various fields of engineering. This technique operates on a microscale and thus uses small volumes. For the encapsulation of bacterial cells – as is the case for our iGEM Project – droplet-based microfluidics is used. As extensively elaborated in the <a href="https://2014.igem.org/Team:TU_Eindhoven/Overview">General Overview Page</a>, the engineered bacteria must be brought in the proximity of PEG polymers. The ultimate goal is to verify the intended function of a cell encapsulation device. Theoretically three processes have to be accomplished:</p>
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                   <p>In short, both membrane proteins had a TAG codon introduced for the non-natural amino acid and a HA-tag for detection methods.  For INPNC the TAG codon and the HA-tag were introduced at the C terminus of the protein (<a href='#Fig1'>Figure 1</a>). The CPX proteins already contained a peptide library part at the N-terminus so the TAG codon was introduced in that part. This has been done by Site Directed Mutagenesis. The HA-tag was introduced at the C-terminus (<a href='#Fig2'>Figure 2</a>). After modification the proteins were renamed to Clickable Outer Membrane Protein x (COMPx), originally CPX, and COMPy, originally INPNC. Protocols for creation of these anchors can be found on the <a href="https://2014.igem.org/Team:TU_Eindhoven/Protocols" >protocol page</a>.</p>
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<img id='Fig1' src="https://static.igem.org/mediawiki/2014/7/75/TU_Eindhoven_COMPy_design.jpg" class="image_wrapper image_fr" width="1085">
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      <li>Combining a PEG solution and a bacterial culture with our engineered <i>E. coli</i> bacteria. These are the essential substances to perform the click reaction. After combining, this is called the Water Phase.</li> 
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<p style="font-size:18px;color:#CCCCCC;">Figure 1. Gene design COMPy (INPNC).</p>
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      <li>The Water Phase has to be dispersed with an Oil Phase into droplets. This oil phase contains a fluorosurfactant in order to prevent aggregation and agglomeration of the droplets. It is crucial to form droplets with one cell in each droplet, since it will assure that each bacterial cell is encapsulated correctly – possible formation of a film is hereby avoided.</li>
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      <li>The result of the previous two processes is a droplet with PEG and engineered E. coli bacteria inside an oil phase. With a microfluidic feature (bumpy mixer) the droplet can be stirred. The droplets are collected in a chamber where the click reaction is initiated with the use of UV light.</li> 
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<img id='Fig2' src="https://static.igem.org/mediawiki/2014/c/cb/TU_Eindhoven_COMPx_design.jpg" class="image_wrapper image_fr" width="1085">
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Before these processes can be performed, certain questions have to be answered and certain research has to be done. For instance, how is it possible to form droplets? What is the optimal method to recollect the content of the droplets? And numerous of other questions that need answering for instance the required flow speed of the water and oil phase and the viscosities of the phases.
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<p style="font-size:18px;color:#CCCCCC;">Figure 2. Gene design COMPy (CPX)</p>
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<img id='Fig1' src="https://static.igem.org/mediawiki/2014/9/97/TU_Eindhoven_Microfluidics_Logo.jpg" class="image_wrapper image_fr" width="1085">
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<p style="font-size:18px;color:#CCCCCC;">Figure 1. TU Eindhoven iGEM 2014 in a microfluidic device.</p>
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<h4>Bibliography</h4>
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<p>Song H, Chen DL, Ismagilov RF. Reactions in droplets in microfluidic channels. Angew Chem Int Ed Engl. 2006;45:7336–7356
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Mazutis, L., Gilbert, J., Ung. W.L., Weitz, D.A., Griffiths, A.D. & Heyman J.A. (2013). Single-cell analysis and sorting using droplet-based microfluidics. Nature, 8(5), pp. 870-91.
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Song H, Chen DL, Ismagilov RF. Reactions in droplets in microfluidic channels. Angew Chem Int Ed Engl. 2006;45:7336–7356
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Latest revision as of 00:54, 18 October 2014

iGEM Team TU Eindhoven 2014

iGEM Team TU Eindhoven 2014

Membrane Anchor Design

In short, both membrane proteins had a TAG codon introduced for the non-natural amino acid and a HA-tag for detection methods. For INPNC the TAG codon and the HA-tag were introduced at the C terminus of the protein (Figure 1). The CPX proteins already contained a peptide library part at the N-terminus so the TAG codon was introduced in that part. This has been done by Site Directed Mutagenesis. The HA-tag was introduced at the C-terminus (Figure 2). After modification the proteins were renamed to Clickable Outer Membrane Protein x (COMPx), originally CPX, and COMPy, originally INPNC. Protocols for creation of these anchors can be found on the protocol page.

Figure 1. Gene design COMPy (INPNC).

Figure 2. Gene design COMPy (CPX)

iGEM Team TU Eindhoven 2014

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