Difference between revisions of "Part:BBa K3402056"

Revision as of 16:18, 27 October 2020

Single-gene editing cassette

This device is composed of 50bp-upPXA1(BBa_K3402037), hph(BBa_K3402012), 50bp-doPXA1(BBa_K3402038), Ptef1(BBa_K3402007), sgPXA1(BBa_K3402039), Tsyn7(BBa_K3402001).

Usage and Biology

We achieve the knockout of PXA1 gene and insert the hygromycin resistant gene as a selection marker. Two parts of PXA1 are homologous arms at the ends. The strongest promoter Ptef1 is used to express sgRNA to guide Cas9 protein to edit PXA1 site.
The transformants were cultured on solid YPD medium with hygromycin. Then the genomes of positive transformants extracted for PCR and gel electrophoresis analysis. As a result, all of the amplified fragments displayed the correct stripe. So all of the target genes were verified to be inserted into the PXA1 site.
The single gene-editing efficiency was 100%.

Comparison of the number of transformants using CRISPR/Cas9 and traditional homologous recombination

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 2171
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 9: / Line 9: @@
 ===Usage and Biology===
-We achieve the knockout of <i>PXA1</i> gene and insert the hygromycin resistant gene as a selection marker. Two parts of <i>PXA1</i> are homologous arms at the ends.
+We achieve the knockout of <i>PXA1</i> gene and insert the hygromycin resistant gene as a selection marker. Two parts of <i>PXA1</i> are homologous arms at the ends. The strongest promoter P<i>tef1</i> is used to express sgRNA to guide Cas9 protein to edit <i>PXA1</i> site.
 <br>
-If modified strains can grow on the plate coated with hygromycin, it is successful to knock <i>PXA1</i> with Cas9 system. By comparison, the editing efficiency of the CRISPR/Cas9 system can be clearly observed.
+The transformants were cultured on solid YPD medium with hygromycin. Then the genomes of positive transformants extracted for PCR and gel electrophoresis analysis. As a result, all of the amplified fragments displayed the correct stripe. So all of the target genes were verified to be inserted into the <i>PXA1</i> site.
 <br>
 The single gene-editing efficiency was 100%.
-[[Image:Single-gene editing cassette-new.png|500px|thumb|center|The plates contained hygromycin]]
+[[Image:Single-gene editing cassette-new.png|500px|thumb|center|Comparison of the number of transformants using CRISPR/Cas9 and traditional homologous recombination]]