Difference between revisions of "Part:BBa K3402058"

Latest revision as of 17:03, 27 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

Based on Double-gene editing expression cassette(BBa_K3402057), we designed the sgRNA that targeted at Leu site. We constructed the Pxa1, GFP, Leu editing expression cassette, and transformed it into the recombinant strain.
The transformants were cultured on solid YPD medium with hygromycin. Then the positive colonies 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 colonies grown on the CM but did not grow on the MM were the correct transformants.

Fig. 1 Green fluorescence observation in fluorescence

Fig. 2 Colony observation a)Complete Medium b)Minimal Medium

The triple gene-editing efficiency was 30%.

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 3: / Line 3: @@
 <partinfo>BBa_K3402058 short</partinfo>
-This device is composed of up-do<i>Leu</i> (BBa_K3402048), sg<i>Leu</i> (BBa_K3402049), 50bp-up<i>PXA1</i> (BBa_K3402037), <i>hph</i> (BBa_K3402012), 50bp-do<i>PXA1</i> (BBa_K3402038), P<i>tef1</i> (BBa_K3402007), sg<i>PXA1</i> (BBa_K3402039), sg<i>GFP</i> (BBa_K3402024), T<i>syn7</i> (BBa_K3402001), up<i>GFP</i> (BBa_K3402025), do<i>GFP</i> (BBa_K3402026).
+This device is composed of up-do<i>Leu</i> ([https://parts.igem.org/Part:BBa_K3402048# BBa_K3402048]), sg<i>Leu</i> ([https://parts.igem.org/Part:BBa_K3402049# BBa_K3402049]), 50bp-up<i>PXA1</i> ([https://parts.igem.org/Part:BBa_K3402037# BBa_K3402037]), <i>hph</i> ([https://parts.igem.org/Part:BBa_K3402012# BBa_K3402012]), 50bp-do<i>PXA1</i> ([https://parts.igem.org/Part:BBa_K3402038# BBa_K3402038]), P<i>tef1</i> ([https://parts.igem.org/Part:BBa_K3402007# BBa_K3402007]), sg<i>PXA1</i> ([https://parts.igem.org/Part:BBa_K3402039# BBa_K3402039]), sg<i>GFP</i> ([https://parts.igem.org/Part:BBa_K3402024# BBa_K3402024]), T<i>syn7</i> ([https://parts.igem.org/Part:BBa_K3402001# BBa_K3402001]), up<i>GFP</i> ([https://parts.igem.org/Part:BBa_K3402025# BBa_K3402025]), do<i>GFP</i> ([https://parts.igem.org/Part:BBa_K3402026# BBa_K3402026]).
 [[Image:Triple-gene editing cassette.png|700px]]
@@ Line 9: / Line 9: @@
 ===Usage and Biology===
+Based on Double-gene editing expression cassette([https://parts.igem.org/Part:BBa_K3402057# BBa_K3402057]), we designed the sgRNA that targeted at Leu site. We constructed the <i>Pxa1</i>, <i>GFP</i>, <i>Leu</i> editing expression cassette, and transformed it into the recombinant strain.
+<br>
+The transformants were cultured on solid YPD medium with hygromycin. Then the positive colonies 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 colonies grown on the CM but did not grow on the MM were the correct transformants.
+[[Image:Triple-gene editing cassette-plate.jpeg|300px|thumb|center|'''Fig. 1''' Green fluorescence observation in fluorescence]]
+[[Image:Colony observation (a CM; b MM).png|500px|thumb|center|'''Fig. 2''' Colony observation '''a)'''Complete Medium '''b)'''Minimal Medium]]
+'''The triple gene-editing efficiency was 30%.'''