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| − | <h4>Parts</h4> | + | <h4>Characterisation of An Existing Part</h4> |
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| + | <p>As our project had a large focus on fluorescence, we decided to characterise a part from the existing iGEM registry that produces red fluorescent protein in order to determine whether we could use it in our project. We decided against using RFP, however here are the results and methods used in the characterisation.</p> |
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| − | Our new parts are all versions of the same modular system, a fusion protein formed from a bacterial transmembrane domain and a zinc-finger domain, with linker domains between domains and a FLAG-tag for immunofluorescence staining. Each transmembrane/zinc-finger domain is independently interchangeable with other transmembrane/zinc-finger domains, though not interchangeable between each other (i.e. the transmembrane domain cannot be swapped for a zinc-finger domain and vice versa).
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| − | <br> The purpose of this fusion protein is to allow bacteria (specifically <i>Escherichia coli</i>) to bind specific short double stranded DNA sequences at their surface; using immobilised DNA the cell should remain bound in place, or this protein can be used for cell identification (barcoding).
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| − | <img src="https://static.igem.org/mediawiki/2015/5/5c/Warwick_Gene_Map.jpeg" align="middle">
| + | <ul> <li> The J04450 gene from the registry was cloned into three different plasmids (pSB1K3, pSB3K3, and pSB4K4) each with a different copy number. </li> |
| − | </p>
| + | <li>Each plasmid was transformed into electrocompetent MG1655-Z1</li> |
| | + | <li>Three colonies of each plasmid were then inoculated in LB and grown overnight.</li> |
| | + | <li>In the morning, each inoculation was refreshed in M9 + casein amino acid (500x dilution), induced with different concentrations of IPTG (0uM, 250uM, and 500uM IPTG) and grown for another 6 hours.</li> |
| | + | <li> They were then transferred into a 96 well plate (200uL per well) and measured their fluorescence every 15 minutes.</li> |
| | + | </ul> |
| | + | </p> |
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| − | | + | <p>The results for our characterisation can be found on the <a href="http://parts.igem.org/Part: BBa_J04450"> J04450 part page in the registry</a >. |
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| − | <p> _______________________________________________________________________________________________________________________________________</p> <h5>Lpp-OmpA-Zif268</h5> | + | <br> |
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| − | This part is our construct with Lpp-OmpA as the transmembrane domain and the Zif268 1AAY fragment as the zinc-finger domain. The transmembrane domain can be exchanged with another using NdeI and AgeI, and the zinc-finger domain can be exchanged using NheI and ClaI. The entire construct is under the pL_lac promoter, and contains a C-terminal FLAG tag.
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| − | </p>
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| − | <a href="http://parts.igem.org/Part:BBa_K1855000">Registry Link</a> | + | <br> |
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| − | <p>_______________________________________________________________________________________________________________________________________</p> <h5>Lpp-OmpA-sZF2</h5>
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| − | This part is our construct with Lpp-OmpA as the transmembrane domain and sZF2 as the zinc-finger domain. The transmembrane domain can be exchanged with another using NdeI and AgeI, and the zinc-finger domain can be exchanged using NheI and ClaI. The entire construct is under the pL_lac promoter, and contains a C-terminal FLAG tag.
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| − | </p>
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| − | <a href="http://parts.igem.org/Part:BBa_K1855001">Registry Link</a>
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| − | <p>_______________________________________________________________________________________________________________________________________</p> <h5>Lpp-OmpA-sZF10</h5>
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| − | This part is our construct with Lpp-OmpA as the transmembrane domain and sZF10 as the zinc-finger domain. The transmembrane domain can be exchanged with another using NdeI and AgeI, and the zinc-finger domain can be exchanged using NheI and ClaI. The entire construct is under the pL_lac promoter, and contains a C-terminal FLAG tag.
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| − | </p>
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| − | <a href="http://parts.igem.org/Part:BBa_K1855002">Registry Link</a>
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| − | <p>_______________________________________________________________________________________________________________________________________</p><h5>Lpp-OmpA-sZF14</h5>
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| − | This part is our construct with Lpp-OmpA as the transmembrane domain and sZF14 as the zinc-finger domain. The transmembrane domain can be exchanged with another using NdeI and AgeI, and the zinc-finger domain can be exchanged using NheI and ClaI. The entire construct is under the pL_lac promoter, and contains a C-terminal FLAG tag.
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| − | </p>
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| − | <a href="http://parts.igem.org/Part:BBa_K1855003">Registry Link</a>
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| − | <p>_______________________________________________________________________________________________________________________________________</p><h5>INP-Zif268</h5>
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| − | This part is our construct with INP as the transmembrane domain and the Zif268 1AAY fragment as the zinc-finger domain. The transmembrane domain can be exchanged with another using NdeI and AgeI, and the zinc-finger domain can be exchanged using NheI and ClaI. The entire construct is under the pL_lac promoter, and contains a C-terminal FLAG tag.
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| − | </p>
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| − | <a href="http://parts.igem.org/Part:BBa_K1855004">Registry Link</a>
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| − | <p>_______________________________________________________________________________________________________________________________________</p> <h5>BclA-Zif268
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| − | </h5>
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| − | This part is our construct with BclA as the transmembrane domain and the Zif268 1AAY fragment as the zinc-finger domain. The transmembrane domain can be exchanged with another using NdeI and AgeI, and the zinc-finger domain can be exchanged using NheI and ClaI. The entire construct is under the pL_lac promoter, and contains a C-terminal FLAG tag.
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| − | </p>
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| − | <a href="http://parts.igem.org/Part:BBa_K1855005">Registry Link</a>
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