From 2007.igem.org

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Contents 1 Preparation of the P1 stock on the w121 strain. 2 Kinetic measurements: 2.1 Growth of DapA- strain (w121) relative to the concentration of DAP in the medium 2.2 Measuring excretion of Dap by MG1655 DapA+ strain in an indirect manner 3 Acinetobacter calcoaceticus ADP1 3.1 Culture 3.2 Medium to triglyceride/wax esters synthesis

Preparation of the P1 stock on the w121 strain.

See protocols. This preparation is necessary for transduction of DapA deletion in MG1655 strain.

Kinetic measurements:

As previously described, we want to measure :

• Growth of DapA- strain (w121) relative to the concentration of DAP in the medium :
• The excretion of Dap by MG1655 DapA+ strain by an indirect way

Growth of DapA- strain (w121) relative to the concentration of DAP in the medium

We measure the kinetic of the w121 as a function of DAP concentration
[DAP] = 5mM

Kinetic Array :Growthing of w121 strain 'supplemented' with S0 (1µL w121 strain incubated ON)
	1	2	3	4	5	6	7	8	9	10	11	12
A
B		200µL LB + 0µL DAP	200µL LB + 2µL DAP	200µL LB + 4µL DAP	200µL LB + 8µL DAP	200µL LB + 12µL DAP
C
D
E
F
G
H

See Results.

Measuring excretion of Dap by MG1655 DapA+ strain in an indirect manner

We measure the growth of DapA- strain in the filtered growth medium of previously incubated MG1655 strain (S0).
S0 = Centrifuged and filtered medium of MG1655 ON
[DAP] = 5mM

Kinetic Array :Growthing of w121 strain 'supplemented' with S0 (1µL w121 strain incubated ON)
	1	2	3	4	5	6	7	8	9	10	11	12
A
B		200µL S0 + 0µL DAP	200µL S0 + 2µL DAP	200µL S0 + 4µL DAP	200µL S0 + 8µL DAP	200µL S0 + 12µL DAP
C
D
E
F
G
H

See Results.

Acinetobacter calcoaceticus ADP1

Culture

We received the strain Acinetobacter calcoaceticus ADP1 from Pasteur Institute (Center of biological resources of the Pasteur Institute): code CIP (ATCC number 33305). The strain was dehydrated and cultured following this protocol on LB agar plate at 30°C overnight.
See protocols

Medium to triglyceride/wax esters synthesis

Acinetobacter ADP1 is able to multiply in LB medium at 30°C. It is also able to synthesise lipids (triglyceride and wax ester) under specific conditions: Low Nitrogen Minimum Medium (LNMM). This medium contains low nitrogen leading the bacterium to decrease its growing. With carbon hydrate source (here succinic acid), the bacteria stock energy into lipids (triglyceride and wax esters).
See protocols
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