Difference between revisions of "Part:BBa K3402058"

Revision as of 05:06, 26 October 2020

Triple-gene editing cassette

This device is composed of up-doLeu (BBa_K3402048), sgLeu (BBa_K3402049), 50bp-upPXA1 (BBa_K3402037), hph (BBa_K3402012), 50bp-doPXA1 (BBa_K3402038), Ptef1 (BBa_K3402007), sgPXA1 (BBa_K3402039), sgGFP (BBa_K3402024), Tsyn7 (BBa_K3402001), upGFP (BBa_K3402025), doGFP (BBa_K3402026).

Usage and Biology

We designed the sgRNA that targeted at Leu site and constructed the PXA1, GFP, Leu triple-gene editing cassette. We transformed it into the recombinant strain. The transformants were cultured on YPD plus hygromycin plates. Then the positive transformants were conducted green fluorescence observation. After that, the positive transformants which lose the function of expressing green fluorescence were dotted on the CM (Complete Medium) and MM (Minimal Medium). The colony which grew on the CM instead of MM were the successfully editing transformants.
The triple gene-editing efficiency was 30%.

Most of transformants lose the function of expressing green fluorescence

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 776
Illegal XhoI site found at 3223
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 174
Illegal NgoMIV site found at 203
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 3859

@@ Line 9: / Line 9: @@
 ===Usage and Biology===
-Based on <i>Pxa1</i>, <i>GFP</i> expression vector, designed the sgRNA that targeted at Leu site. We construct the <i>Pxa1</i>, <i>GFP</i>, <i>Leu</i> editing expression cassette below, and transform it into the recombinant strain.
+We designed the sgRNA that targeted at <i>Leu</i> site and constructed the <i>PXA1</i>, <i>GFP</i>, <i>Leu</i> triple-gene editing cassette. We transformed it into the recombinant strain. The transformants were cultured on YPD plus hygromycin plates. Then the positive transformants were conducted green fluorescence observation. After that, the positive transformants which lose the function of expressing green fluorescence were dotted on the CM (Complete Medium) and MM (Minimal Medium). The colony which grew on the CM instead of MM were the successfully editing transformants.
-<br>
-The transformants were cultured on YPD plus hygromycin plates. Then the positive transformants were conducted green fluorescence observation. After that, the positive transformants without fluorescence were dotted on the Complete Medium (CM) and Minimal Medium (MM). Then the colony grown on the Complete Medium but did not grow on the Minimal Medium were the right transformants.
 <br>
 The triple gene-editing efficiency was 30%.