Difference between revisions of "Part:BBa K4335003"
Line 5: Line 5:
 
We used the Rbcs2 promoter and terminator, and since the Rbcs2 Promotor Rhizobium-derived transcription factor was low, we added the UTR of the heat shock promoter HSP70A Promotor to enhance the promoter and thus the heterologous expression of the Staygold fluorescent protein
 
We used the Rbcs2 promoter and terminator, and since the Rbcs2 Promotor Rhizobium-derived transcription factor was low, we added the UTR of the heat shock promoter HSP70A Promotor to enhance the promoter and thus the heterologous expression of the Staygold fluorescent protein
 
StayGold, a green fluorescent protein (GFP) from the jellyfish family Endocystis, was chosen as our reporter fluorescent protein because it is orders of magnitude more photostable than any currently available fluorescent protein, and because the excitation and emission wavelengths of Staygold do not overlap with the chloroplast fluorescence of Chlamydomonas reinhardtii itself.
 
StayGold, a green fluorescent protein (GFP) from the jellyfish family Endocystis, was chosen as our reporter fluorescent protein because it is orders of magnitude more photostable than any currently available fluorescent protein, and because the excitation and emission wavelengths of Staygold do not overlap with the chloroplast fluorescence of Chlamydomonas reinhardtii itself.
 +
<html>
 +
 +
 +
 +
<h2>Usage</h2>
 +
We inserted ChlamyHgR into plasmids pTX2038 and pTX2040 as marker genes for transformation screening.<br>
 +
<br>
 +
<h2>Result</h2>
 +
<h3>Plasmid construction</h3>
 +
To verify our successful assembly, we designed two primers, Hgy-F and Hgy-R, for PCR verification. The primer location, primer sequence and gel map are shown in the following figure:<br>
 +
<br>
 +
    <figure>
 +
        <img src="https://static.igem.wiki/teams/4335/wiki/m.png" width="100%" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rem">
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
    <figure>
 +
        <img src="https://static.igem.wiki/teams/4335/wiki/mcherry-3.png" width="100%" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rem">mCherry and the position of primer targeting
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
    <figure>
 +
        <img src="https://static.igem.wiki/teams/4335/wiki/mcherry-4.png" width="20%" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rem">Electrophoregram of amplification products,M is DNA Marker.
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
 +
<h3>Validation of conversion results</h3>
 +
 +
    <figure>
 +
        <img src="https://static.igem.wiki/teams/4335/wiki/mcherry-5.jpg" width="100%" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rem">Fluorescence excitation of positive clones transferred into pTX2038 vector compared with wild type mCherry, together with DIC field and chlorophyll excitation as fluorescence control.
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
 +
<h2>Reference</h2>
 +
 +
</html>
  
  

Revision as of 19:27, 10 October 2022


ChlamyHgR

We used the Rbcs2 promoter and terminator, and since the Rbcs2 Promotor Rhizobium-derived transcription factor was low, we added the UTR of the heat shock promoter HSP70A Promotor to enhance the promoter and thus the heterologous expression of the Staygold fluorescent protein StayGold, a green fluorescent protein (GFP) from the jellyfish family Endocystis, was chosen as our reporter fluorescent protein because it is orders of magnitude more photostable than any currently available fluorescent protein, and because the excitation and emission wavelengths of Staygold do not overlap with the chloroplast fluorescence of Chlamydomonas reinhardtii itself.

Usage

We inserted ChlamyHgR into plasmids pTX2038 and pTX2040 as marker genes for transformation screening.

Result

Plasmid construction

To verify our successful assembly, we designed two primers, Hgy-F and Hgy-R, for PCR verification. The primer location, primer sequence and gel map are shown in the following figure:

mCherry and the position of primer targeting

Electrophoregram of amplification products,M is DNA Marker.

Validation of conversion results

Fluorescence excitation of positive clones transferred into pTX2038 vector compared with wild type mCherry, together with DIC field and chlorophyll excitation as fluorescence control.

Reference


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 782
    Illegal NgoMIV site found at 890
  • 1000
    COMPATIBLE WITH RFC[1000]