Difference between revisions of "Part:BBa K4905016"
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<h2>Usage and Biology</h2> | <h2>Usage and Biology</h2> | ||
<p> | <p> | ||
| − | This part was made to obtain a hydrophobic fluorescent protein that could be used for FRAP imaging experiments to characterize part <a style="color:#F6B227" href="https://parts.igem.org/Part:BBa_K4905006">BBa_K4905006</a> | + | This part was made to obtain a hydrophobic fluorescent protein that could be used for FRAP imaging experiments to characterize part <a style="color:#F6B227" href="https://parts.igem.org/Part:BBa_K4905006">BBa_K4905006</a>. It consists of the fluorescent protein mNeonGreen <a style="color:#F6B227" href="https://parts.igem.org/Part:BBa_K1761003">BBa_K1761003</a> and a hydrophobic Elastin-Like Polypeptide sequence <a style="color:#F6B227" href="https://parts.igem.org/Part:BBa_K4905003">BBa_K4905003</a>.</p> |
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| + | <h2>Characterization</h2> | ||
| + | The part was cloned into a pBAD vector as evidenced by a double digestion, with restriction enzymes cutting at both ends of the gene fragment (Figure 1). | ||
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| + | <div style="width:100%; text-align:center;"> | ||
| + | <img src="https://static.igem.wiki/teams/4905/wiki/mneongreen-g.png" style="width:20vw;" class="results-img enlarge-image" alt="Image 12"><br> | ||
| + | </div> | ||
| + | <figcaption style="text-align:center; font-size: 12px;"><b>Figure 1:</b><i> Double digestion of pBad vector containing [A3G2]<sub>12</sub>-mNeonGreen</i></figcaption><br> | ||
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| + | Using this vector, [A3G2]<sub>12</sub>-mNeonGreen was expressed in <i>E. coli </i> BL21(DE3). Evidence of expression can be seen in Figure 2, where the protein was loaded on an SDS-PAGE gel before and after purification with inverse transition cycling. In addition, the cell pellet showed fluorescence when excited with a blue light. | ||
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| + | <div style="width:100%; text-align:center;"> | ||
| + | <img src="https://static.igem.wiki/teams/4905/wiki/co-expression-mng-z1z2-black.png" style="width: 25vw ;" class="results-img enlarge-image" alt="Image 13"> | ||
| + | <img src="https://static.igem.wiki/teams/4905/wiki/bba-k4905006/img-2903.jpeg" style="width: 20vw" class="results-img enlarge-image" alt="Image 14"> | ||
| + | </div | ||
| + | <figcaption style="text-align:center; font-size: 12px;"><b>Figure 2:</b><i> A) SDS-PAGE gel showing the co-expressed proteins (left three lanes) versus a negative control in which no arabinose was added and therefore no expression of [A3G2]<sub>12</sub>-mNeonGreen was induced (right three lanes). B) Bacterial cell pellet excited by a blue light transilluminator showing the fluorescence emitted by mNeonGreen. | ||
| + | </i></figcaption><br> | ||
| + | |||
| + | |||
</body> | </body> | ||
</html> | </html> | ||
Latest revision as of 11:28, 11 October 2023
mneongreen with elastin-like polypeptide
Sequence and features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This part was made to obtain a hydrophobic fluorescent protein that could be used for FRAP imaging experiments to characterize part BBa_K4905006. It consists of the fluorescent protein mNeonGreen BBa_K1761003 and a hydrophobic Elastin-Like Polypeptide sequence BBa_K4905003.
Characterization
The part was cloned into a pBAD vector as evidenced by a double digestion, with restriction enzymes cutting at both ends of the gene fragment (Figure 1).
Using this vector, [A3G2]12-mNeonGreen was expressed in E. coli BL21(DE3). Evidence of expression can be seen in Figure 2, where the protein was loaded on an SDS-PAGE gel before and after purification with inverse transition cycling. In addition, the cell pellet showed fluorescence when excited with a blue light.
