Difference between revisions of "Part:BBa K4335009"

Revision as of 18:41, 10 October 2022 (view source) Hang Zhang (Talk | contribs) ← Older edit		Latest revision as of 19:02, 10 October 2022 (view source) Hang Zhang (Talk | contribs)
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	<br>		<br>
	<figure>		<figure>
−	<img src="https://static.igem.wiki/teams/4335/wiki/zhanghang-1.png" width="30%" style="float:center">	+	<img src="https://static.igem.wiki/teams/4335/wiki/zhanghang-1.png" width="60%" style="float:center">
	<figcaption>		<figcaption>
−	<p style="font-size:1rem">	+	<p style="font-size:1rem">sgRNA
	</p>		</p>
	</figcaption>		</figcaption>
	</figure>		</figure>
	<h2>Result</h2>		<h2>Result</h2>
−	<h3>Plasmid construction</h3>	+	The following is the PCR verification electrophoresis diagram after we assembled various sgRNAs in Golden Gate Assembly.
−	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/zhanghang-insert-site-grna-scaffold.jpg" width="100%" style="float:center">
−	<figure>	+
−	<img src="https://static.igem.wiki/teams/4335/wiki/m.png" width="100%" style="float:center">	+
	<figcaption>		<figcaption>
−	<p style="font-size:1rem">	+	<p style="font-size:1rem">Gel run of samples from colony PCR.Primer length ~660 bp. M: 2000 bp DNA Marker; 38: pTX2038 vector; 40: pTX2040 vector; sgRNA1-3: different sgRNAs is designed.
	</p>		</p>
	</figcaption>		</figcaption>
	</figure>		</figure>
−	<figure>	+	Figure 1 shows that the colony PCR has successfully amplified about 660 bp bands, which correspond to the vector corresponding to the sgRNA of each target gene (pTX2038/pTX2040). It preliminarily indicates that we have successfully inserted the sgRNA of the target gene into the vector (pTX2038/pTX2040), and then Sanger sequencing of each insert site has been carried out, which proves that we have successfully constructed.<br>
−	<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>		<br>
−	[2]Parenteau, J. et al. Introns are mediators of cell response to starvation. Nature 565, 612–617 (2019).	+	<a href="">collection</a>
	</html>		</html>

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Latest revision as of 19:02, 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

The following is the PCR verification electrophoresis diagram after we assembled various sgRNAs in Golden Gate Assembly.

Figure 1 shows that the colony PCR has successfully amplified about 660 bp bands, which correspond to the vector corresponding to the sgRNA of each target gene (pTX2038/pTX2040). It preliminarily indicates that we have successfully inserted the sgRNA of the target gene into the vector (pTX2038/pTX2040), and then Sanger sequencing of each insert site has been carried out, which proves that we have successfully constructed.

collection

Sequence and Features