Difference between revisions of "Part:BBa K3506050"
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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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[[Image:T--BNU-China--N19.JPG|200px|thumb|center|Figure. 1 Experimental group (4500FOA with gRNA and Cas9)]]
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[[Image:T--BNU-China--N19.JPG|200px|thumb|left|Figure. 1 Experimental group (4500FOA with gRNA and Cas9)]]
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[[Image:T--BNU-China--4500.JPG|200px|thumb|center|Figure. 2 Control group (4500FOA)]]
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[[Image:T--BNU-China--4500.JPG|200px|thumb|right|Figure. 2 Control group (4500FOA)]]
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 07:46, 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.

Properties

We electroporated this part and spCas9(BBa_K2130013) into Cryptococcus neoformans 4500FOA as experimental group. Use 4500FOA as control group gRNA was designed to target the ADE2 gene. A loss-of-function mutation in ADE2 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 Cryptococcus neoformans.

Figure. 1 Experimental group (4500FOA with gRNA and Cas9)
Figure. 2 Control group (4500FOA)

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.