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| | <h3>Marburg 2018 improved this part</h3> | | <h3>Marburg 2018 improved this part</h3> |
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| − | Here, we present the improvement of an iGEM-part for (a mostly faster,) easier and cheaper selection which suits our needs.</p> | + | Here, we present the improvement of an iGEM-part for easier and cheaper selection which suits our needs.</p> |
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| | iGEM provides the universal acceptor plasmid BBa_P10500 for creating new PhytoBricks. This plasmid contained the <i>lacZ-α</i> part in the cloning sites for blue-white screening. As we wanted to establish <i>Vibrio natriegens</i> as a chassis for cloning, we investigated if the wild type strain is compatible with blue-white screening. Unfortunately, cloning using BBa_P10500 in <i>V. natriegens</i> shows no difference between colonies containing the <i>lacZ-α</i> dropout and those without. | | iGEM provides the universal acceptor plasmid BBa_P10500 for creating new PhytoBricks. This plasmid contained the <i>lacZ-α</i> part in the cloning sites for blue-white screening. As we wanted to establish <i>Vibrio natriegens</i> as a chassis for cloning, we investigated if the wild type strain is compatible with blue-white screening. Unfortunately, cloning using BBa_P10500 in <i>V. natriegens</i> shows no difference between colonies containing the <i>lacZ-α</i> dropout and those without. |
| − | To overcome this limitation and to enable fast and reliable cloning with <i>V. natriegens</i>, we decided to establish a new visualization method. Our improved part is a derivative of the iGEM BBa_P10500 containing a sfGFP dropout as fluorescent selection marker. <p></p> | + | To overcome this limitation and to enable fast and reliable cloning with <i>V. natriegens</i>, we decided to establish a new visualization method. Our improved part, namely <a href=" https://parts.igem.org/Part:BBa_K2560002 "> |
| | + | <abbr title=" Link to the iGEM part registry "> |
| | + | BBa_K2560002</abbr></a>, is a derivative of the iGEM BBa_P10500 containing a sfGFP dropout as fluorescent selection marker. <p></p> |
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| − | <html> | + | </html> |
| | <div style="width:100%;display:flex;flex-direction:row;flex-wrap: wrap; justify-content:space-evenly; align-items:center;"> | | <div style="width:100%;display:flex;flex-direction:row;flex-wrap: wrap; justify-content:space-evenly; align-items:center;"> |
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| − | [[File:T--Marburg--K2560002.png|800px|thumb|left]] | + | [[File:T--Marburg--K2560002.png|400px|thumb|left|'''Figure 1''': <b> Plasmidmap of BBa_K2560002 </b> <br> Our improved part BBa_K2560002 is a derivate of the BBa_P10500 containing sfGFP drop out.]] |
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| | </div> | | </div> |
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| | <p> | | <p> |
| − | In the pictures 2, you can see the wild type <i>V. natriegens</i> in comparison with the iGEM BBa_P10500 forming white colonies as well as bright green colonies containing the improved part BBa_K2560002. No differences between the wild type and the BBa_P10500 containing colonies are noticable. On the contrary, our new BBa_K2560002 part leads to a strong visual distinction to colonies which do not possess the sfGFP. In this way, we created a part for universal LVL0 cloning with a improved selection without the need of additional supplements like Xgal or IPTG. | + | In the pictures 2, you can see the wild type <i>V. natriegens</i> in comparison with the iGEM BBa_P10500 forming white colonies as well as bright green colonies containing the improved part <a href=" https://parts.igem.org/Part:BBa_K2560002 "> |
| | + | <abbr title=" Link to the iGEM part registry "> |
| | + | BBa_K2560002</abbr></a>. No differences between the wild type and the BBa_P10500 containing colonies are noticable. On the contrary, our new <a href=" https://parts.igem.org/Part:BBa_K2560002 "> |
| | + | <abbr title=" Link to the iGEM part registry "> |
| | + | BBa_K2560002</abbr></a> part leads to a strong visual distinction to colonies which do not possess the sfGFP. In this way, we created a part for universal LVL0 cloning with a improved selection without the need of additional supplements like Xgal or IPTG. |
| | </p><p> | | </p><p> |
| − | Our part not only suits for <i>V. natriegens</i> but is convenient for the frequently used cloning host <i>E. coli</i>. As it can be seen in the picture 3, <i>E. coli</i> containing the sfGFP possess a considerable strong green colour even without the use of UV light and is just or even more distinguishable from the wild type as the <i>lacZ</i> containing blue colonies. By using our improved part BBa_K2560002 instead of the iGEM part BBa_P10500 work and money for the addition of the required supplements can be saved and the risk of not functional plates for selection is decreased. In our experiments, we were using 40 μg per Liter Xgal and 0,5 mM IPTG. Calculating with current prices, 100 plates supplemented with Xgal andIPTG costs about 80 dollar. </p><p> | + | Our part not only suits for <i>V. natriegens</i> but is convenient for the frequently used cloning host <i>E. coli</i>. As it can be seen in the picture 3, <i>E. coli</i> containing the sfGFP possess a considerable strong green colour even without the use of UV light and is just or even more distinguishable from the wild type as the <i>lacZ</i> containing blue colonies. By using our improved part <a href=" https://parts.igem.org/Part:BBa_K2560002 "> |
| | + | <abbr title=" Link to the iGEM part registry "> |
| | + | BBa_K2560002</abbr></a> instead of the iGEM part BBa_P10500 work and money for the addition of the required supplements can be saved and the risk of not functional plates for selection is decreased. In our experiments, we were using 40 μg per Liter Xgal and 0,5 mM IPTG. Calculating with current prices, 100 plates supplemented with <a href=" https://www.applichem.com/shop/produktdetail/as/x-gal-ibiochemicai/ "> |
| | + | <abbr title=" link to shop "> |
| | + | Xgal</abbr></a> and |
| | + | <a href=" https://www.carlroth.com/de/de/Chemikalien/A-Z-Chemikalien/I/IPTG/IPTG/p/000000010000911300020023_de "> |
| | + | <abbr title=" link to shop "> |
| | + | IPTG</abbr></a> costs about 80 dollar. </p><p> |
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| | </html> | | </html> |
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| | [[File:T--Marburg--E.coli+V.natriegens.png|800px|thumb|left]] | | [[File:T--Marburg--E.coli+V.natriegens.png|800px|thumb|left]] |
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| − | </div> | + | </div> |
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| − | <p>By using our improved part for cloning, the detection of successfully ligated clones from non is feasible for strains which are not compatible with blue-white screening and therefor becomes more universal, faster and cheaper than before. </p> | + | <br>By using our improved part for cloning, the detection of successfully ligated clones from non is feasible for strains which are not compatible with blue-white screening and therefor becomes more universal, faster and cheaper than before. |
This is the universal acceptor plasmid for creating new PhytoBricks parts. It contains the lacZ-alpha fragment in the cloning site for blue-white screening through the use of IPTG and X-Gal on the agar plates.
