Paris/July 24

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Revision as of 09:09, 25 July 2007

Minipreps

BBa_E0422 in pSB1A2 - Clone 1&2 - MP6.1' & MP6.2'
BBa_E0241 in pSB1A2 - Clone 1 - MP7.1'
BBa_J61047 in pSB1A2 - Clone 1&2 - MP9.1' & MP9.2'

Growth kinetics of w121 strain

We make an array to test growth of w121 on different growth media (LB, S0.2, S0.4, S0.6, S0.8), supplemented with different amount of DAP.

Two questions are addressed by the following assay:

-What is the growth behaviour of w121 (dapA- strain) at different concentrations of DAP? -How does w121 strain grow on filtrates of MG1655 growth medium; that is, does MG1655 secrete DAP during growth?

MG1655 was grown on LB medium and the growth medium was filtered free of bacteria at different DO (Optical Densities) during exponential growth phase:

S0.2 (at DO=0.2) S0.4 (at DO=0.4) S0.6 S0.8

W121 was grown on different media & Growth kinetics were measured:

LB line B in the array S0.2 (at DO=0.2) line C in the array S0.4 line D in the array S0.6 line E & F in the array S0.8 line G in the array

In the different columns, DAP was added to the indicated final concentrations (without taking into account DAP produced by MG1655 regarding the recycled growth media)

Kinetic Array :w121 kinetic as a function of DAP and supplemented medium (S0.x)
	1	2	3	4	5	6	7	8	9	10	11	12
A	H₂0	H₂0	H₂0	H₂0	H₂0	H₂0	H₂0	H₂0	H₂0	H₂0	H₂0	H₂0
B	H₂0	LB+0µM DAP	LB+20µM DAP	LB+25µM DAP	LB+30µM DAP	LB+35µM DAP	LB+40µM DAP	LB+45µM DAP	LB+50µM DAP	LB+55µM DAP	LB+60µM DAP	H₂0
C	H₂0	S0.2+0µM DAP	S0.2+5µM DAP	S0.2+10µM DAP	S0.2+15µM DAP	S0.2+20µM DAP	S0.2+25µM DAP	S0.2+30µM DAP	S0.2+35µM DAP	S0.2+40µM DAP	S0.2+45µM DAP	H₂0
D	H₂0	S0.4+0µM DAP	S0.4+5µM DAP	S0.4+10µM DAP	S0.4+15µM DAP	S0.4+20µM DAP	S0.4+25µM DAP	S0.4+30µM DAP	S0.4+35µM DAP	S0.4+40µM DAP	S0.4+45µM DAP	H₂0
E	H₂0	S0.6+0µM DAP	S0.6+2µM DAP	S0.6+5µM DAP	S0.6+8µM DAP	S0.6+11µM DAP	S0.6+14µM DAP	S0.6+17µM DAP	S0.6+20µM DAP	S0.6+23µM DAP	S0.6+26µM DAP	H₂0
F	H₂0	S0.6+0µM DAP	S0.6+2µM DAP	S0.6+5µM DAP	S0.6+8µM DAP	S0.6+11µM DAP	S0.6+14µM DAP	S0.6+17µM DAP	S0.6+20µM DAP	S0.6+23µM DAP	S0.6+26µM DAP	H₂0
G	H₂0	S0.8+0µM DAP	S0.8+2µM DAP	S0.8+5µM DAP	S0.8+8µM DAP	S0.8+11µM DAP	S0.8+14µM DAP	S0.8+17µM DAP	S0.8+20µM DAP	S0.8+23µM DAP	S0.8+26µM DAP	H₂0
H	H₂0	H₂0	H₂0	H₂0	H₂0	H₂0	H₂0	H₂0	H₂0	H₂0	H₂0	H₂0

Ligation & transformation reactions

Ligations
Number	Insert	Insert Volume	Vector	Vector Volume	Comments
L1	D23 (AraC/pBad promoter FI)		D9 (J61002 ready for insertion of a FI)
L2	D18 (BB dig. lox66DapAE.coli)		D22 (pSB1A2 Eco, Pst)
L3	D19 (BB dig. lox66DapAsubtilis)		D22 (pSB1A2 Eco, Pst)

To do:

D23 (AraC/pBad promoter FI) in D9 (J61002 ready for insertion of a FI)
D18 (BB dig. lox66DapAE.coli) & D19 (BB dig. lox66DapAsubtilis) in D22 (pSB1A2 Eco, Pst)
D20 (Lox66-DapAE.coli BI) & D21 (lox66-DapAsubtilis BI) in D1 (pJ23100 BV) & D3 (pJ23107 BV)
D14 (Cre ORF) in D15 (B0030 BV)
D27 (Lox71-ftsZ BI) in D1 (pJ23100 BV) & D3 (pJ23107 BV)
lox66 (annealing O14 & O15) & lox71 (annealing O16 & O17) in D22 (pSB1A2 Eco+Pst)

Annealing of Lox66 and Lox71

Lox66 and Lox71 oligos were designed to form a double-stranded DNA. The extremities bear cohesive overhangs corresponding to digestion by EcoRI and SpeI. See oligos 14 + 15 and 16 + 17 here.

Annealing mix:

8 μL of each of the concentrated primers
4 μL of salt solution (10 mM NaCl)
20 μL of water

Mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally.
More information [http://openwetware.org/wiki/Annealing_complementary_primers here]

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+|width=25%| Vector
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 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L1
-|D9 (J61002 ready for insertion of a FI)
-|
 |D23 (AraC/pBad promoter FI)
+|
+|D9 (J61002 ready for insertion of a FI)
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