Difference between revisions of "Part:BBa K4335002"

 
 
(8 intermediate revisions by the same user not shown)
Line 3: Line 3:
 
<partinfo>BBa_K4335002 short</partinfo>
 
<partinfo>BBa_K4335002 short</partinfo>
  
−
Here we present StayGold, a green fluorescent protein (GFP) derived from the jellyfish Cytaeis uchidae. StayGold is over one order of magnitude more photostable than any currently available fluorescent protein and has a cellular brightness similar to mNeonGreen. Researchers used StayGold to image the dynamics of the endoplasmic reticulum (ER) with high spatiotemporal resolution over several minutes using structured illumination microscopy (SIM) and observed substantially less photobleaching than with a GFP variant optimized for stability in the ER. StayGold will substantially reduce the limitations imposed by photobleaching, especially in the live cell or volumetric imaging. We chose StayGold as our reporter fluorescent protein because it has an order of magnitude greater photostability than any currently available fluorescent protein, and the excitation and emission wavelengths of Staygold do not overlap with the chloroplast fluorescence of Chlamydomonas reinhardtii itself
+
<html>
 +
StayGold, a green fluorescent protein (GFP) , fluoresces in expression. StayGold has an order of magnitude more photostability than any currently available fluorescent protein. Commonly used to label and trace cellular components. BBa_K4335002 is our StayGold gene optimized for the codon of Chlamydomonas reinhardtii.
 +
<h2>Additional Materials</h2>
 +
StayGold was extracted from <i>Cytaeis uchidae </i> by Atsushi Miyawaki et al. by the RIKEN Center for Brain Science Research on April 25, 2022.  
 +
 
 +
<figure>
 +
        <img src="https://static.igem.wiki/teams/4335/wiki/staygoldfzx0.png" width="100%" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rem">Absorption spectrum and emission spectrum of StayGold.<a href="https://www.fpbase.org/protein/staygold/">[Image origin]</a>
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
<figure>
 +
        <img src="https://static.igem.wiki/teams/4335/wiki/staygoldfzx1.png" width="75%" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rem">StayGold and other 15 kinds of fluorescent protein fluorescence intensity changes with time
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
 
 +
StayGold has an order of magnitude more light stability than any currently available fluorescent protein and has a cell brightness similar to that of mNeonGreen. Compared with the 15 fluorescent proteins, StayGold showed good photostability.
 +
 
 +
<h2>Usage</h2>
 +
We introduced the StayGold gene into the <a href="https://parts.igem.org/Part:BBa_K4335040">[pTX2040]</a> plasmid.
 +
PCrRBCS2 is used as the promoter and TCrRBCS2 is used as the terminator to achieve the expression of the StayGold gene.
 +
<h2>Result</h2>
 +
<h3>Plasmid construction</h3>
 +
We designed two primers: Staygold-F and Staygold-R, and confirmed the successful assembly by PCR amplification.
 +
<figure>
 +
        <img src="https://static.igem.wiki/teams/4335/wiki/staygoldfzx4.png" width="100%" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rem">
 +
        </p>The primers
 +
        </figcaption>
 +
    </figure>
 +
 
 +
<figure>
 +
        <img src="https://static.igem.wiki/teams/4335/wiki/staygoldfzx2.png" width="100%" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rem">Primers targeted location
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
 
 +
<figure>
 +
        <img src="https://static.igem.wiki/teams/4335/wiki/staygoldfzx3.png" width="20%" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rem">The electrophoretogram of StayGold after amplification
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
 
 +
<h3>Functional Identification</h3>
 +
 
 +
<figure>
 +
        <img src="https://static.igem.wiki/teams/4335/wiki/staygoldfzx5.jpg" width="100%" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rem">Fluorescence excitation of positive clones transferred into pTX2040 vector compared with wild-type mCherry, together with DIC field and chlorophyll excitation as fluorescence controls.
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
<h2>Reference</h2>
 +
[1] Hirano, M. et al. A highly photostable and bright green fluorescent protein. Nat. Biotechnol. 40, 1132–1142 (2022).
 +
</html>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 08:22, 11 October 2022


StayGold, a newly discoverd green fluorescent protein

StayGold, a green fluorescent protein (GFP) , fluoresces in expression. StayGold has an order of magnitude more photostability than any currently available fluorescent protein. Commonly used to label and trace cellular components. BBa_K4335002 is our StayGold gene optimized for the codon of Chlamydomonas reinhardtii.

Additional Materials

StayGold was extracted from Cytaeis uchidae by Atsushi Miyawaki et al. by the RIKEN Center for Brain Science Research on April 25, 2022.

Absorption spectrum and emission spectrum of StayGold.[Image origin]

StayGold and other 15 kinds of fluorescent protein fluorescence intensity changes with time

StayGold has an order of magnitude more light stability than any currently available fluorescent protein and has a cell brightness similar to that of mNeonGreen. Compared with the 15 fluorescent proteins, StayGold showed good photostability.

Usage

We introduced the StayGold gene into the [pTX2040] plasmid. PCrRBCS2 is used as the promoter and TCrRBCS2 is used as the terminator to achieve the expression of the StayGold gene.

Result

Plasmid construction

We designed two primers: Staygold-F and Staygold-R, and confirmed the successful assembly by PCR amplification.

The primers

Primers targeted location

The electrophoretogram of StayGold after amplification

Functional Identification

Fluorescence excitation of positive clones transferred into pTX2040 vector compared with wild-type mCherry, together with DIC field and chlorophyll excitation as fluorescence controls.

Reference

[1] Hirano, M. et al. A highly photostable and bright green fluorescent protein. Nat. Biotechnol. 40, 1132–1142 (2022).

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 540
  • 1000
    COMPATIBLE WITH RFC[1000]