Test pLasR/cI - Revision history

XiZhu at 14:54, 11 July 2008

2008-07-11T14:54:58Z

XiZhu at 14:53, 11 July 2008

2008-07-11T14:53:22Z

XiZhu at 14:42, 11 July 2008

2008-07-11T14:42:37Z

XiZhu at 14:53, 1 July 2008

2008-07-01T14:53:05Z

XiZhu at 14:51, 1 July 2008

2008-07-01T14:51:45Z

AnGordon at 20:03, 30 June 2008

2008-06-30T20:03:57Z

New page

pLasR/cI is constitutively off and is repressed by cI. The addition of p-AI-1, activates LasR causing the promoter to turn on. The addition of cI to should repress the promoter causing it to turn off.

{| border="1" cellpadding="2"
!width="30"|LasR
!width="30"|cI
!width="30"|p-AI-1 + GFP
!width="30"|p-AI-1 - GFP
|-
| 0 || 0 || 0 || 0
|-
| 1 || 0 || 1 || 0
|-
| 0 || 1 || 0 || 0
|-
| 1 || 1 || 0 || 0
|}

@@ Line 23: / Line 23: @@
 I13453+B0034+C0079+B0015
-plasR/cI→RBS+GFP
+plasR/cI→RBS+GFP+tt
 K09110?+E0240               does not glow
@@ Line 33: / Line 33: @@
 I13453+B0034+C0079+B0015
-plasR/cI→RBS+GFP
+plasR/cI→RBS+GFP+tt
 K09110?+E0240               glows
@@ Line 39: / Line 39: @@
 '''Ligation plan for test sequences'''
 Digest plasR/cI with S/P, E0240 with X/P; do ligation.

@@ Line 36: / Line 36: @@
 K09110?+E0240               glows
@@ Line 44: / Line 43: @@
 K09110? ligated with E0240
-Digest RBS with S/P, E0240 with X/P; do ligation.
+Digest plasR/cI with S/P, E0240 with X/P; do ligation.

@@ Line 16: / Line 16: @@
 |}
-plasR/cI→RBS+GFP does not glow
+'''Test pLasR/cI:'''
-K09110?+E0240
+pBAD→RBS+LasR+TT (from James DC)
-) Test LasR activation:
+plasR/cI→RBS+GFP
-pBAD→RBS+LasR+t+t+plsrA/cI→RBS+GFP does not glow
+K09110?+E0240               does not glow
-I13453+B0034+C0079+B0015+K09110?+E0240
+'''add P-AI-1'''
-add P-AI-1
+pBAD→RBS+LasR+TT (from James DC)
-pBAD→RBS+LsrR+t+t+plsrA/cI→RBS+GFP glows
+I13453+B0034+C0079+B0015
-I13453+B0034+C0079+B0015+K091106+E0240
+plasR/cI→RBS+GFP
-pBAD→RBS+cI+TT+plasR/cI→GFP does not glow
+'''Ligation plan for test sequences'''
 K09110? ligated with E0240
-need to ligate some promoter (digest with E/S) with an RBS (digest E/X) and ligate lasR(digest with E/S) with a double terminator (digest with E/X)
+Digest RBS with S/P, E0240 with X/P; do ligation.
-the 2) test sequence should glow and the 4) should not.
+Digest plasR/cI with S/P, RBS+E0240 with X/P; do ligation.

← Older revision		Revision as of 14:53, 1 July 2008
Line 38:		Line 38:
	3) Test cI repression:		3) Test cI repression:

−	~~pBAD→RBS+cI+TT+plasR/cI→GFP glows~~
−
−	~~I13453+B0034+C0051+B0015+K09110?+E0240~~
−
−	~~add cI protein~~

	pBAD→RBS+cI+TT+plasR/cI→GFP does not glow		pBAD→RBS+cI+TT+plasR/cI→GFP does not glow

← Older revision		Revision as of 14:51, 1 July 2008
Line 15:		Line 15:
	\| 1 \|\| 1 \|\| 0 \|\| 0		\| 1 \|\| 1 \|\| 0 \|\| 0
	\|}		\|}
		+
		+	1)Control group
		+
		+	plasR/cI→RBS+GFP does not glow
		+
		+	K09110?+E0240
		+
		+
		+	2) Test LasR activation:
		+
		+	pBAD→RBS+LasR+t+t+plsrA/cI→RBS+GFP does not glow
		+
		+	I13453+B0034+C0079+B0015+K09110?+E0240
		+
		+	add P-AI-1
		+
		+	pBAD→RBS+LsrR+t+t+plsrA/cI→RBS+GFP glows
		+
		+	I13453+B0034+C0079+B0015+K091106+E0240
		+
		+
		+	3) Test cI repression:
		+
		+	pBAD→RBS+cI+TT+plasR/cI→GFP glows
		+
		+	I13453+B0034+C0051+B0015+K09110?+E0240
		+
		+	add cI protein
		+
		+	pBAD→RBS+cI+TT+plasR/cI→GFP does not glow
		+
		+	I13453+B0034+C0051+B0015+K09110?+E0240
		+
		+
		+	4) Test lasR activation VS cI repression :
		+
		+	pBAD→RBS+lasR+cI+TT+plsrA/cI→GFP does not glows
		+
		+	I13453+B0034+C0079+C0051+B0015+K09110?+E0240
		+
		+	add P-AI-1
		+
		+	pBAD→RBS+lsrR+cI+TT+plsrA/cI→GFP does not glows
		+
		+	I13453+B0034+C0079+C0051+B0015+K09110?+E0240
		+
		+
		+
		+	Ligation plan for three test sequences
		+
		+	1)
		+
		+	K09110? ligated with E0240
		+
		+	need to ligate some promoter (digest with E/S) with an RBS (digest E/X) and ligate lasR(digest with E/S) with a double terminator (digest with E/X)
		+
		+
		+	2)
		+
		+	K09110? ligated with E0240
		+
		+	need to ligate some promoter (digest with E/S) with an RBS (digest E/X) and ligate lasR(digest with E/S) with a double terminator (digest with E/X)
		+
		+	then ligate the promoter+RBS to the 5' end of the lasR+TT parts together
		+
		+	then ligate that part 5' to the plasR/cI+GFP
		+
		+
		+	3)
		+
		+	ligate cI to the double terminator
		+
		+	the ligate the promoter+RBS from the first test sequence to the 5' end of the cI+TT
		+
		+	ligate this to the 5' end of the plasR/cI+GFP
		+
		+
		+	4)
		+
		+	ligate the cI+TT from the second test sequence to the 3' end of the lasR coding region
		+
		+	then ligate the promoter+RBS from the first test sequence to the 5' end of that
		+
		+	next ligate the plasA/cI to the 3' end of that entire sequence
		+
		+
		+	the 2) and 4) test sequences will also need to be tested by adding P-AI-1
		+
		+	the 2) test sequence should glow and the 4) should not.