Paris/August 7

From 2007.igem.org

< Paris(Difference between revisions)

Latest revision as of 17:54, 7 October 2007

Study of the pBAD promoter

We've put the AraC-pBAD promoter (BBa_I0500) in the expression vector BBa_J61002 (between EcoRI and SpeI). After overnight culture of E.coli transformed by this plamid, we diluted 1/100 and waited 2 hours. At OD=0.2, we divided the culture in 2 subcultures, one with arabinose (final 0.2%) and one without.

1 hour later, we took pictures with fluorescence microscope (triplicate):

- the first line represent subculture without arabinose induction. - the second represent subculture with 0.2% arabinose induction.

5 hours later:

Interpretation:

Arabinose induction looks to work. mRFP expression looks to increase with time (the acquisition and analyse were identical at each time).

Digestion reactions

Digestion Products
Number	Product Name	Matrix Name	Enzyme 1	Enzyme 2	Size	Description
D37'.1	lox71-gfp(tripart) BI	L12.1 miniprep product (from 30/07 ligation reaction)	XbaI	PstI		lox71_RBS-rfp-double terminator extracted as a backward insert
D39'	BI-insert lox66-DapAE.coli	lox66-DapAE.coli in pTOPO (P5 PCR product)(clones 1+2)	XbaI	PstI		lox66-dapAcoli extracted for usage as a backward insert
D41	araC/pBad-J61002 plasmid SP dig.	L1.2, L1".1 & L1".2 miniprep product	SpeI	PstI		J61002 plasmid containing araC/pBad promotor digested for insertion of a BI CDS

Minipreps

Number	Name	Description
L1.2		S16.2
MPT3.1		pTOPO P6.1
MPT3.2		pTOPO P6.7
MPT4.1		pTOPO P8.2
MPT4.2		pTOPO P8.5
MPT4.3		pTOPO P8.7
MPT4.4		pTOPO P8.8
MPT4.5		pTOPO P8.10
MPT5.1		pTOPO P9.1
MPT5.2		pTOPO P9.2
MPT5.3		pTOPO P9.4

@@ Line 1: / Line 1: @@
+[[Paris/August 6|yesterday]] -- [[Paris/August 8|tomorrow]] <br>
 == Study of the pBAD promoter ==
-We've put the AraC-pBAD promoter (<bbpart>BBa_I0500</bbpart>) in the expression vector <bbpart>BBa_J61002</bbpart> (between EcoRI and SpeI)
+We've put the AraC-pBAD promoter (<bbpart>BBa_I0500</bbpart>) in the expression vector <bbpart>BBa_J61002</bbpart> (between EcoRI and SpeI). After overnight culture of E.coli transformed by this plamid, we diluted 1/100 and waited 2 hours. At OD=0.2, we divided the culture in 2 subcultures, one with arabinose (final 0.2%) and one without.
+* '''1 hour''' later, we took pictures with fluorescence microscope (triplicate): <br>
+- the first line represent subculture without arabinose induction.
+- the second represent subculture with 0.2% arabinose induction.
+[[Image: t0_pBad-AraC.jpg|center|600px]]<br>
+* '''5 hours''' later:
+[[Image: t5_pBad-AraC.jpg|center|600px]]
+* '''Interpretation:'''
+Arabinose induction looks to work. mRFP expression looks to increase with time (the acquisition and analyse were identical at each time).
 ==  Digestion reactions  ==
@@ Line 64: / Line 77: @@
 |width=25%| Name
 |width=50%| Description
-|width=15%| Products in use
 |- style="background: #cccccc;"
-| style="background: #ccffcc;" |MPT2
+| style="background: #ccffcc;" |L1.2
 |
 |S16.2
-|
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |MPT3.1
 |
 |pTOPO P6.1
-|
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |MPT3.2
 |
 |pTOPO P6.7
-|
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |MPT4.1
 |
 |pTOPO P8.2
-|
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |MPT4.2
 |
 |pTOPO P8.5
-|
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |MPT4.3
 |
 |pTOPO P8.7
-|
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |MPT4.4
 |
 |pTOPO P8.8
-|
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |MPT4.5
 |
 |pTOPO P8.10
-|
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |MPT5.1
 |
 |pTOPO P9.1
-|
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |MPT5.2
 |
 |pTOPO P9.2
-|
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |MPT5.3
 |
 |pTOPO P9.4
-|
 |}