Difference between revisions of "Part:BBa K4335009"

 
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The transcript of gRNA scaffold can bind to SpCas9. The Insert site can be inserted into the sgRNA targeting specific target genes through the Golden Gate assembly.
 
The transcript of gRNA scaffold can bind to SpCas9. The Insert site can be inserted into the sgRNA targeting specific target genes through the Golden Gate assembly.
 +
<html>
 +
<h2>Usage</h2>
 +
Template of sgRNA. Use Golden Gate (BsaⅠ) to replace the template sequence with a ~20bp guide sequence. Add 'ACTT' on the 5'-terminal of your guide sequence and 'AAAC' on the reverse 5'-terminal.<br>
 +
<br>
 +
  <figure>
 +
        <img src="https://static.igem.wiki/teams/4335/wiki/zhanghang-1.png" width="30%" style="float:center">
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        <figcaption>
 +
        <p style="font-size:1rem">
 +
        </p>
 +
        </figcaption>
 +
  </figure>
 +
<h2>Result</h2>
 +
<h3>Plasmid construction</h3>
 +
To validate the mCherry sequence in our vector, we designed mCherry-f and mCherry-R primers to verify our successful assembly.
 +
 +
<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>Functional Identification</h3>
 +
We introduced the mCherry-containing plasmid pTX2038 into <i>Chlamydomonas reinhardtii</i> by electroporation and observed it using Fluorescence microscope.
 +
<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>
 +
[1] Codon usage and tRNA content in unicellular and multicellular organisms. T Ikemura. Mol Biol Evol (1985) 2 (1): 13-34.<br>
 +
<br>
 +
[2]Parenteau, J. et al. Introns are mediators of cell response to starvation. Nature 565, 612–617 (2019).
 +
</html>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 18:41, 10 October 2022


Insert site+gRNA scaffold

The transcript of gRNA scaffold can bind to SpCas9. The Insert site can be inserted into the sgRNA targeting specific target genes through the Golden Gate assembly.

Usage

Template of sgRNA. Use Golden Gate (BsaⅠ) to replace the template sequence with a ~20bp guide sequence. Add 'ACTT' on the 5'-terminal of your guide sequence and 'AAAC' on the reverse 5'-terminal.

Result

Plasmid construction

To validate the mCherry sequence in our vector, we designed mCherry-f and mCherry-R primers to verify our successful assembly.

mCherry and the position of primer targeting

Electrophoregram of amplification products,M is DNA Marker.

Functional Identification

We introduced the mCherry-containing plasmid pTX2038 into Chlamydomonas reinhardtii by electroporation and observed it using Fluorescence microscope.

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

[1] Codon usage and tRNA content in unicellular and multicellular organisms. T Ikemura. Mol Biol Evol (1985) 2 (1): 13-34.

[2]Parenteau, J. et al. Introns are mediators of cell response to starvation. Nature 565, 612–617 (2019).

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 25
    Illegal BsaI.rc site found at 7