Team:TU Eindhoven/Design/Plasmid Design

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                   <h2>Membrane Anchor Design</h2>
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                   <h2>Plasmid Design</h2>
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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 using 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.</p>
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                   <p>The chosen vector for expression is pET29a. At both anchors at the N- and C-terminus the restriction sites for NdeI and SacI were introduced to clone the genes into a pET29a vector. The modified genes were first digested with the restriction enzymes and so was the pET29a vector. (<a href='#Fig1'>Figure 1</a> and <a href='#Fig2'>Figure 2</a>) After digestion the genes were ligated into the vector.</p>
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<img id='Fig1' src="https://static.igem.org/mediawiki/2014/4/4b/TU_Eindhoven_COMPy_design.png" class="image_wrapper image_fr" width="1085">
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<img id='Fig1' src="https://static.igem.org/mediawiki/2014/1/1e/TU_Eindhoven_PET29a_COMPx.png" class="image_wrapper image_fr" width="1085">
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<p style="font-size:18px;color:#CCCCCC;">Figure 1. Gene design COMPy (INPNC).</p>
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<p style="font-size:18px;color:#CCCCCC;">Figure 1. Plasmid  design pET29a(+) COMPx</p>
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<img id='Fig2' src="https://static.igem.org/mediawiki/2014/4/43/TU_Eindhoven_COMPx_design.png" class="image_wrapper image_fr" width="1085">
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<img id='Fig2' src="https://static.igem.org/mediawiki/2014/2/21/TU_Eindhoven_PET29a_COMPy.png" class="image_wrapper image_fr" width="1085">
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<p style="font-size:18px;color:#CCCCCC;">Figure 2. Gene design COMPy (CPX)</p>
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<p style="font-size:18px;color:#CCCCCC;">Figure 2. Plasmid design pET29a(+) COMPy</p>
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Revision as of 20:28, 9 October 2014

iGEM Team TU Eindhoven 2014

iGEM Team TU Eindhoven 2014

Plasmid Design

The chosen vector for expression is pET29a. At both anchors at the N- and C-terminus the restriction sites for NdeI and SacI were introduced to clone the genes into a pET29a vector. The modified genes were first digested with the restriction enzymes and so was the pET29a vector. (Figure 1 and Figure 2) After digestion the genes were ligated into the vector.

Figure 1. Plasmid design pET29a(+) COMPx

Figure 2. Plasmid design pET29a(+) COMPy

iGEM Team TU Eindhoven 2014

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