Paris/July 24

From 2007.igem.org

(Difference between revisions)

Revision as of 10:19, 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'

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 here

@@ Line 4: / Line 4: @@
 <bbpart>BBa_E0241</bbpart> in <bbpart>pSB1A2</bbpart> - Clone 1 - MP7.1'<br>
 <bbpart>BBa_J61047</bbpart> in <bbpart>pSB1A2</bbpart> - Clone 1&2 - MP9.1' & MP9.2'<br>
-== 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)
-{{Paris_KineticArray| Title = w121 kinetic as a function of DAP and supplemented medium (S0.x)
-|A1=H<sub>2</sub>0|A2=H<sub>2</sub>0|A3=H<sub>2</sub>0|A4=H<sub>2</sub>0
-|A5=H<sub>2</sub>0|A6=H<sub>2</sub>0|A7=H<sub>2</sub>0|A8=H<sub>2</sub>0
-|A9=H<sub>2</sub>0|A10=H<sub>2</sub>0|A11=H<sub>2</sub>0|A12=H<sub>2</sub>0
-|B1=H<sub>2</sub>0
-|B2=LB+0µM DAP|B3=LB+20µM DAP|B4=LB+25µM DAP|B5=LB+30µM DAP|B6=LB+35µM DAP
-|B7=LB+40µM DAP|B8=LB+45µM DAP|B9=LB+50µM DAP|B10=LB+55µM DAP|B11=LB+60µM DAP
-|B12=H<sub>2</sub>0|C1=H<sub>2</sub>0
-|C2=S0.2+0µM DAP|C3=S0.2+5µM DAP|C4=S0.2+10µM DAP|C5=S0.2+15µM DAP|C6=S0.2+20µM DAP
-|C7=S0.2+25µM DAP|C8=S0.2+30µM DAP|C9=S0.2+35µM DAP|C10=S0.2+40µM DAP|C11=S0.2+45µM DAP
-|C12=H<sub>2</sub>0|D1=H<sub>2</sub>0
-|D2=S0.4+0µM DAP|D3=S0.4+5µM DAP|D4=S0.4+10µM DAP|D5=S0.4+15µM DAP|D6=S0.4+20µM DAP
-|D7=S0.4+25µM DAP|D8=S0.4+30µM DAP|D9=S0.4+35µM DAP|D10=S0.4+40µM DAP|D11=S0.4+45µM DAP
-|D12=H<sub>2</sub>0|E1=H<sub>2</sub>0
-|E2=S0.6+0µM DAP|E3=S0.6+2µM DAP|E4=S0.6+5µM DAP|E5=S0.6+8µM DAP|E6=S0.6+11µM DAP
-|E7=S0.6+14µM DAP|E8=S0.6+17µM DAP|E9=S0.6+20µM DAP|E10=S0.6+23µM DAP|E11=S0.6+26µM DAP
-|E12=H<sub>2</sub>0|F1=H<sub>2</sub>0
-|F2=S0.6+0µM DAP|F3=S0.6+2µM DAP|F4=S0.6+5µM DAP|F5=S0.6+8µM DAP|F6=S0.6+11µM DAP
-|F7=S0.6+14µM DAP|F8=S0.6+17µM DAP|F9=S0.6+20µM DAP|F10=S0.6+23µM DAP|F11=S0.6+26µM DAP
-|F12=H<sub>2</sub>0|G1=H<sub>2</sub>0
-|G2=S0.8+0µM DAP|G3=S0.8+2µM DAP|G4=S0.8+5µM DAP|G5=S0.8+8µM DAP|G6=S0.8+11µM DAP
-|G7=S0.8+14µM DAP|G8=S0.8+17µM DAP|G9=S0.8+20µM DAP|G10=S0.8+23µM DAP|G11=S0.8+26µM DAP
-|G12=H<sub>2</sub>0|H1=H<sub>2</sub>0|H2=H<sub>2</sub>0|H3=H<sub>2</sub>0|H4=H<sub>2</sub>0
-|H5=H<sub>2</sub>0|H6=H<sub>2</sub>0|H7=H<sub>2</sub>0|H8=H<sub>2</sub>0
-|H9=H<sub>2</sub>0|H10=H<sub>2</sub>0|H11=H<sub>2</sub>0|H12=H<sub>2</sub>0}}
 == Ligation & transformation reactions ==