Difference between revisions of "Part:BBa K4905016"
 
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<h2>Usage and Biology<h/2>
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<h2>Usage and Biology</h2>
 
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This part was made to obtain a hydrophobic fluorescent protein that could be used for FRAP imaging experiments. 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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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>
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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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<img src="https://static.igem.wiki/teams/4905/wiki/mneongreen-g.png" style="width:20vw;" class="results-img enlarge-image" alt="Image 12"><br>
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<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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<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">
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<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">
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<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.
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</i></figcaption><br>
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 11:28, 11 October 2023


mneongreen with elastin-like polypeptide

Sequence and features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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).
Image 12
Figure 1: Double digestion of pBad vector containing [A3G2]12-mNeonGreen

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.
Image 13 Image 14
Figure 2: 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]12-mNeonGreen was induced (right three lanes). B) Bacterial cell pellet excited by a blue light transilluminator showing the fluorescence emitted by mNeonGreen.