From 2007.igem.org

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 2.3 Acinetobacter calcoaceticus ADP1 culture

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 as a function of DAP concentration (1µL w121 strain incubated ON)
	1	2	3	4	5	6	7	8	9	10	11	12
A											{{{A11}}}	{{{A12}}}
B		200µL LB + 0µL DAP	200µL LB + 2µL DAP	200µL LB + 4µL DAP	200µL LB + 8µL DAP	200µL LB + 12µL DAP					{{{B11}}}	{{{B12}}}
C											{{{C11}}}	{{{C12}}}
D											{{{D11}}}	{{{D12}}}
E											{{{E11}}}	{{{E12}}}
F											{{{F11}}}	{{{F12}}}
G											{{{G11}}}	{{{G12}}}
H	{{{H1}}}	{{{H2}}}	{{{H3}}}	{{{H4}}}	{{{H5}}}	{{{H6}}}	{{{H7}}}	{{{H8}}}	{{{H9}}}	{{{H10}}}	{{{H11}}}	{{{H12}}}

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											{{{A11}}}	{{{A12}}}
B		200µL S0 + 0µL DAP	200µL S0 + 2µL DAP	200µL S0 + 4µL DAP	200µL S0 + 8µL DAP	200µL S0 + 12µL DAP					{{{B11}}}	{{{B12}}}
C											{{{C11}}}	{{{C12}}}
D											{{{D11}}}	{{{D12}}}
E											{{{E11}}}	{{{E12}}}
F											{{{F11}}}	{{{F12}}}
G											{{{G11}}}	{{{G12}}}
H	{{{H1}}}	{{{H2}}}	{{{H3}}}	{{{H4}}}	{{{H5}}}	{{{H6}}}	{{{H7}}}	{{{H8}}}	{{{H9}}}	{{{H10}}}	{{{H11}}}	{{{H12}}}

See Results.

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Acinetobacter calcoaceticus ADP1 culture

We received the strain Acinetobacter calcoaceticus ADP1 from Pasteur Intitute (Center of biological ressources of the Pasteur Institute): code CIP 104273 (ATCC number 33305). The strain was dehydrated and cultured following this protocole on LB agar plate at 30°C overnight: