Difference between revisions of "Part:BBa K3506050"
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<b><font size="3">Properties</font></b>
 
<b><font size="3">Properties</font></b>
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We electroporated this part and spCas9(BBa_K2130013) into <i>Cryptococcus neoformans</i> 4500FOA as experimental group. Use 4500FOA as control group gRNA was designed to target the <i>ADE2</i> gene. A loss-of-function mutation in <i>ADE2</i> results in an adenine auxotroph that forms pink colonies on culture plates containing a low level of adenine, therefore enabling a visual evaluation of the action of CRISPR-Cas9. In our result, pink colonies grew on the YNBA plates, indicating that gRNA successful targeted at the ADE2 locus in <i>Cryptococcus neoformans</i>.
 
We electroporated this part and spCas9(BBa_K2130013) into <i>Cryptococcus neoformans</i> 4500FOA as experimental group. Use 4500FOA as control group gRNA was designed to target the <i>ADE2</i> gene. A loss-of-function mutation in <i>ADE2</i> results in an adenine auxotroph that forms pink colonies on culture plates containing a low level of adenine, therefore enabling a visual evaluation of the action of CRISPR-Cas9. In our result, pink colonies grew on the YNBA plates, indicating that gRNA successful targeted at the ADE2 locus in <i>Cryptococcus neoformans</i>.
  
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1.Construct recombinant plasmid. Insert spacer sequence of gRNA on PRH003 plasmid.  
 
1.Construct recombinant plasmid. Insert spacer sequence of gRNA on PRH003 plasmid.  
 +
 
2.Transform the product (2.5μL) into DH5α competent cells（50μL）, grow cells on LB-amphenicol medium. Incubate at 37°C overnight. Monoclones are selected by colony PCR. Expanding culture colonies at 37℃ 200rpm, then extracting plasmids and sequencing.  
 
2.Transform the product (2.5μL) into DH5α competent cells（50μL）, grow cells on LB-amphenicol medium. Incubate at 37°C overnight. Monoclones are selected by colony PCR. Expanding culture colonies at 37℃ 200rpm, then extracting plasmids and sequencing.  
 +
 
3.Use Kpn1 enzyme to linearise the plasmids and transform them into <i>Cryptococcus neoformans</i> 4500FOA by electroporation.   
 
3.Use Kpn1 enzyme to linearise the plasmids and transform them into <i>Cryptococcus neoformans</i> 4500FOA by electroporation.   
 +
 
4.The <i>C. neoformans<i> is spread on YNBA selection medium, and the transformants grow after being cultured in an incubator kept at 30℃ for 4 days. Then the culture is transferred to a refrigerator at 4℃.  
 
4.The <i>C. neoformans<i> is spread on YNBA selection medium, and the transformants grow after being cultured in an incubator kept at 30℃ for 4 days. Then the culture is transferred to a refrigerator at 4℃.  
 +
 
5.Red colonies are selected and inoculated into YPD medium, then place in an incubator kept at 30℃ for 4 days. Finally it is kept at 4℃ refrigerator.  
 
5.Red colonies are selected and inoculated into YPD medium, then place in an incubator kept at 30℃ for 4 days. Finally it is kept at 4℃ refrigerator.  
 +
 
6.Comparing the experimental group with the control group, we evaluate whether this system work well according to the color of colonies.
 
6.Comparing the experimental group with the control group, we evaluate whether this system work well according to the color of colonies.

Revision as of 06:43, 27 October 2020


gRNA targets ADE2 gene

A guide RNA (gRNA) is an artificial RNA which is designed to bind a certain DNA sequence. It can combine with Cas9 protein to play a role in the cleavage of target DNA. We design a gRNA that targeted on ADE2 gene to specifically knock this gene in Cryptococcus neoformans.

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]



Expenrimental approach

1.Construct recombinant plasmid. Insert spacer sequence of gRNA on PRH003 plasmid.

2.Transform the product (2.5μL) into DH5α competent cells（50μL）, grow cells on LB-amphenicol medium. Incubate at 37°C overnight. Monoclones are selected by colony PCR. Expanding culture colonies at 37℃ 200rpm, then extracting plasmids and sequencing.

3.Use Kpn1 enzyme to linearise the plasmids and transform them into Cryptococcus neoformans 4500FOA by electroporation.

4.The C. neoformans<i> is spread on YNBA selection medium, and the transformants grow after being cultured in an incubator kept at 30℃ for 4 days. Then the culture is transferred to a refrigerator at 4℃.

5.Red colonies are selected and inoculated into YPD medium, then place in an incubator kept at 30℃ for 4 days. Finally it is kept at 4℃ refrigerator.

6.Comparing the experimental group with the control group, we evaluate whether this system work well according to the color of colonies.