Paris/July 24

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[[Paris/July 23|yesterday]] -- [[Paris/July 25|tomorrow]] <br>
==Minipreps==
==Minipreps==
-
<bbpart>BBa_E0422</bbpart> in <bbpart>pSB1A2</bbpart> - Clone 1&2 - MP6.1' & MP6.2'<br>
+
<bbpart>BBa_E0422</bbpart> in <bbpart>pSB1A2</bbpart> - Clone 1&2 - [[Paris/Freezer#Plasmids:_MiniPrep_Products|MP6.1' & MP6.2']]<br>
-
<bbpart>BBa_E0241</bbpart> in <bbpart>pSB1A2</bbpart> - Clone 1 - MP7.1'<br>
+
<bbpart>BBa_E0241</bbpart> in <bbpart>pSB1A2</bbpart> - Clone 1 - [[Paris/Freezer#Plasmids:_MiniPrep_Products|MP7.1']]<br>
-
<bbpart>BBa_J61047</bbpart> in <bbpart>pSB1A2</bbpart> - Clone 1&2 - MP9.1' & MP9.2'<br>
+
<bbpart>BBa_J61047</bbpart> in <bbpart>pSB1A2</bbpart> - Clone 1&2 - [[Paris/Freezer#Plasmids:_MiniPrep_Products|MP9.1' & MP9.2']]<br>
-
== Growth kinetics of w121 strain ==
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== Ligation & transformation reactions ==
-
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.
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{|
 +
|- style="background: #ccccff;"
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! colspan="5" style="background: #ccccff;" | Ligations
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|- style="background: #ccccff; text-align: center;"
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|width=5%| Number
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|width=25%| Insert
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|width=5%| Insert Volume
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|width=25%| Vector
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|width=5%| Vector Volume
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|width=35%| Comments
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L1
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|D23 (AraC/pBad promoter FI)
 +
|
 +
|D9 (J61002 ready for insertion of a FI)
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L2
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|D18 (BB dig. lox66DapAE.coli)
 +
|
 +
|D22 (pSB1A2 Eco, Pst)
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L3
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|D19 (BB dig. lox66DapAsubtilis)
 +
|
 +
|D22 (pSB1A2 Eco, Pst)
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L4
 +
|D20 (Lox66-DapAE.coli BI)
 +
|
 +
|D1 (pJ23100 BV)
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L5
 +
|D21 (lox66-DapAsubtilis BI)
 +
|
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|D1 (pJ23100 BV)
 +
|
 +
|
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|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L6
 +
|D20 (Lox66-DapAE.coli BI)
 +
|
 +
|D3 (pJ23107 BV)
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L7
 +
|D21 (lox66-DapAsubtilis BI)
 +
|
 +
|D3 (pJ23107 BV)
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L8
 +
|D14 (Cre ORF)
 +
|
 +
|D15 (B0030 BV)
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L9
 +
|D27 (Lox71-ftsZ BI)
 +
|
 +
|D1 (pJ23100 BV)
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L10
 +
|D27 (Lox71-ftsZ BI)
 +
|
 +
|D3 (pJ23107 BV)
 +
|
 +
|
 +
|}
-
Two questions are addressed by the following assay:
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*lox66 (annealing O14 & O15) & lox71 (annealing O16 & O17) in D22 (pSB1A2 Eco+Pst)
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-What is the growth behaviour of w121 (dapA- strain) at different concentrations of DAP?
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== Annealing of Lox66 and Lox71 ==
-
-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:
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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 [[Paris/Oligos|here]].
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S0.2 (at DO=0.2) 
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'''Annealing mix:'''
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S0.4 (at DO=0.4)
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* 8 μL of each of the concentrated primers
-
S0.6                       
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* 4 μL of salt solution (10 mM NaCl)
-
S0.8                       
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* 20 μL of water
-
+
-
W121 was grown on different media & Growth kinetics were measured:
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Mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally.<br>
 +
More information [http://openwetware.org/wiki/Annealing_complementary_primers here]
 +
==Purification of PCR Products==
-
 
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* RBS-DapAColi = P8
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LB                          line B in the array
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* RBS-DapASubtilis = P9
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S0.2 (at DO=0.2)            line C in the array
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* Lox71-FtsA-FtsZ = P10
-
S0.4                        line D in the array
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* DGAT1 = P3
-
S0.6                        line E & F in the array
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* DGAT2 = P4
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S0.8                        line G in the array
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-
 
+
-
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)
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-
 
+
-
 
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-
{{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
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-
|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
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|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 ==
+
-
 
+
-
To do:
+
-
*D23 (AraC/pBad promoter FI) in D9 (J61002 ready for insertion of a FI)
+
-
*D18 & D19 in D22
+
-
*
+

Latest revision as of 17:50, 7 October 2007

yesterday -- tomorrow

Contents

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)
L4 D20 (Lox66-DapAE.coli BI) D1 (pJ23100 BV)
L5 D21 (lox66-DapAsubtilis BI) D1 (pJ23100 BV)
L6 D20 (Lox66-DapAE.coli BI) D3 (pJ23107 BV)
L7 D21 (lox66-DapAsubtilis BI) D3 (pJ23107 BV)
L8 D14 (Cre ORF) D15 (B0030 BV)
L9 D27 (Lox71-ftsZ BI) D1 (pJ23100 BV)
L10 D27 (Lox71-ftsZ BI) 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]

Purification of PCR Products

  • RBS-DapAColi = P8
  • RBS-DapASubtilis = P9
  • Lox71-FtsA-FtsZ = P10
  • DGAT1 = P3
  • DGAT2 = P4
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