Difference between revisions of "Part:BBa K3506050"
Chenyiling (Talk | contribs) |
|||
| Line 3: | Line 3: | ||
<partinfo>BBa_K3506050 short</partinfo> | <partinfo>BBa_K3506050 short</partinfo> | ||
| − | A | + | 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 <i>ADE2</i> gene to specifically knock this gene in <i>Cryptococcus neoformans</i>. |
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
| + | 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. | ||
| + | |||
| + | <b><font size="3">Properties</font></b> | ||
| + | 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>. | ||
| + | |||
| + | Figure 1. Experimental group (4500FOA with gRNA and Cas9) | ||
| + | |||
| + | Fifure 2. Control group (4500FOA) | ||
| + | |||
<!-- --> | <!-- --> | ||
| Line 17: | Line 26: | ||
<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>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. | ||
Revision as of 06:42, 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.