Difference between revisions of "Part:BBa K3506050"
 
(19 intermediate revisions by 4 users not shown)
Line 3: Line 3:
 
<partinfo>BBa_K3506050 short</partinfo>
 
<partinfo>BBa_K3506050 short</partinfo>
  
−
A small guide RNA (sgRNA) component created by fusing an approximately 20 nt target-sequence RNA (crRNA) to a tailored bacterial tracrRNA. We design the sgRNA targeted on <i>ADE2</i> to specifically knock out this gene in <i>Cryptococcus neoformans</i>.
+
A guide RNA (gRNA) is an artificial RNA that is designed to bind a certain DNA sequence. It can combine with Cas9 protein to play a role in the cleavage of targeting DNA. We design a gRNA that targets <i>ADE2</i> gene to specifically knock this gene in <i>Cryptococcus neoformans</i>.
 +
 
 +
<b><font size="3">Properties</font></b>
 +
 
 +
We electroporated this part and spCas9 ([https://parts.igem.org/Part:BBa_K2130013 BBa_K2130013]) into <i>Cryptococcus neoformans</i> 4500FOA as the experimental group. Use 4500FOA as the 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 (Figure 1), indicating that gRNA was successfully targeted the <i>ADE2</i> locus in <i>Cryptococcus neoformans</i>.
 +
[[Image:T--BNU-China--N19 &amp; 4500.JPG|500px|thumb|center|Figure 1. Left: the experimental group (4500FOA with gRNA and Cas9); Right: the control group (4500FOA)]]
 +
 
 +
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
 +
 +
 +
  
 
<!-- -->
 
<!-- -->
Line 17: Line 27:
 
<partinfo>BBa_K3506050 parameters</partinfo>
 
<partinfo>BBa_K3506050 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
 +
<b><font size="3">Expenrimental approach</font></b>
 +
 +
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 <i>Cryptococcus neoformans</i> 4500FOA by electroporation. 
 +
 +
4.The <i>Cryptococcus 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.Pink colonies are selected and inoculated into YPD medium, then placed 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.

Latest revision as of 23:35, 27 October 2020


gRNA targets ADE2 gene

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

Properties

We electroporated this part and spCas9 (BBa_K2130013) into Cryptococcus neoformans 4500FOA as the experimental group. Use 4500FOA as the 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 (Figure 1), indicating that gRNA was successfully targeted the ADE2 locus in Cryptococcus neoformans.

Figure 1. Left: the experimental group (4500FOA with gRNA and Cas9); Right: the 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 Cryptococcus neoformans 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.Pink colonies are selected and inoculated into YPD medium, then placed 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.