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===Usage and Biology===
 
===Usage and Biology===
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<b><font size="3">Biology and Usage</font></b>
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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 when using gRNA targets on <i>ADE2</i> gene and Cas9 protein together, we can evaluate whether this system work well according to the color of colonies.
 
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 when using gRNA targets on <i>ADE2</i> gene and Cas9 protein together, we can evaluate whether this system work well according to the color of colonies.
  

Revision as of 06:44, 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.