Team:Grenoble/Biology/Protocols/AND test

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iGEM Grenoble 2012

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"AND" gate test

Goal

Measure the dependence of the activity of the pBAD promoter on the concentrations of cAMP and arabinose.

Procedure

Outline

The day before: Grow BW25113 cya- containing the paraBAD_RBS_GFP plasmid of Alon (plasmid collection) in LB + Kanamycin. Leave on the shaker at 37°C overnight.
Measure expression in different concentrations of arabinose and cAMP in M9 complemented with glucose, acetate or glycerol.

Protocol

Outline

Add 10 µL of an appropriate mix of cAMP and arabinose to each well of a 96-well microplate (see plate layout below). Add 130 µL of M9 into each well. The medium is prepared fresh, but not filtered. Then, add 10 µL of the overnight culture and read in Fusion at 37°C.

cAMP: 0 to 3.2 mM: 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 mM (2-fold dilutions)

arabinose: 0 to 0.1%: 0.1, 0.02, 0.004, 0.0008, 0.00016, 0 % (5-fold dilutions)


Details

Make the cAMP gradient by serial dilution of a 100 mM solution

  1. 10 µL of 100 mM cAMP into all wells of row A.
  2. 5 µL of H2O into all wells, except row A.
  3. Transfer 5 µL sequentially from row A to row B, then B to C, etc. until row G. Mix at each step.
  4. Remove 5 µL from row G.

Make the arabinose gradient by serial dilution

  1. 20 µL of 3% arabinose into wells A1 to H1 of a new microplate.
  2. Transfer 10 µL to column 2.
  3. Add 40 µL to column 2, mix, and transfer 10 µL to column 3.
  4. Continue until column 5.
  5. 10 µL of H2O to column 6.
  6. Transfer 5 µL of column 1 to columns 1 and 2 of the reading plate.
  7. Continue until column 6 of the arabinose plate. This column goes in duplicate into columns 11 and 12 of the reading plate.

Add M9 medium

To each well, add 130 µL of M9. Preparation see below. The medium was not filtered and the solutions were not sterile.
Remove bubbles from the wells using a toothpick.

Add bacteria last

Start the reaction by adding 10 µL of the overnight culture to each well of the microplate.
Start reading in the Fusion at 37°C (The instrument has been prewarmed).

Plate layout



    1    
    2    
    3    
    4    
    5    
    6    
    7    
    8    
    9    
   10   
   11   
   12   

   A   
3.2
1
3.2
1
3.2
0.2
3.2
0.2
3.2
0.04
3.2
0.04
3.2
0.008
3.2
0.008
3.2
0.002
3.2
0.002
3.2
0
3.2
0

   B   
1.6
1.6
1.6
1.6
1.6
1.6
1.6
1.6
1.6
1.6
1.6
1.6

   C   
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8

   D   
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4

   E   
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2

   F   
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1

   G   
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05

   H   
0
0
0
0
0
0
0
0
0
0
0
0

The first number is the final concentration of cAMP in mM, the second the final concentration of arabinose in per mille.
The arabinose concentrations are the same in the entire row (only the first row is shown).

Retrieved from "http://2012.igem.org/Team:Grenoble/Biology/Protocols/AND_test"