Biology Reference
In-Depth Information
FBA, by setting the growth rate to the value of the dilution rate used in experiments
and subsequently minimizing for consumption of source of respective element (suc-
cinate, ammonia, phosphate, and sulfate).
Computational Methods
The model was created and maintained using ToBiN (Toolbox for Biochemical Net-
works, http://www.lifewizz.com). The optimizations (FBA, FVA, OptKnock) were
computed by free, open source, solvers from the COIN-OR family (COmputational
INfrastructure for Operations Research, http://www.coin-or.org) or by the lp_solve
version 5.5 (http://lpsolve.sourceforge.net/5.5/) software package. All computations
were performed on a Personal Computer with a Intel Core 2 2.40 GHz CPU and 2GB
of RAM.
Experimental Methods
Media and Chemicals
Pseudomonas
putida
KT2440 was grown either on EM-medium (Bacto Trypton - 20
g, Yeast-Extract - 5 g, NaCl - 5 g, Glucose 0.5%, H
2
O
dist
at 1,000 ml; the glucose was
as 10% solution autoclaved separately and added in appropriate amount) or SOC-
medium (Bacto Trypton - 2%, Yeast-extract - 0.5%, Glucose - 20 nM, NaCl - 10 mM,
KCl - 2.5 mM, MgCl
2
- 10 mM, MgSO
4
- 10 mM, H
2
O
dist
ad 1,000 ml; magnesium
salts were autoclaved separately and subsequently merged with the remaining compo-
nents) or minimal medium (10×; Na
2
HPO
4
- 50 g, KH
2
PO
4
- 100 g, MgSO
4
×7H
2
O - 2
g, (NH
4
)
2
SO
4
- 20 g, CaCl
2
- 0.01 g, FeSO
4
× 7H
2
O - 0.01 g, H
2
O
dist
ad 1,000 ml; the
potassium and sodium salts were dissolved separately and subsequently mixed with
other dissolved salts; pH was set to 7.0 by adding 10 mM NaOH) with different com-
pounds as the sole carbon source.
BIOLOG Substrate Utilization Experiments
Pseudomonas putida
KT2440 was tested for its ability to utilize various carbon sourc-
es using BIOLOG GN2 Microplates [31] (BIOLOG Inc. Hayward, CA, USA). All
procedures were performed as indicated by the manufacturer. Bacteria were grown
overnight in 28°C on a BIOLOG Universal Growth agar plate. Afterwards they were
swabbed from the surface of the plate and suspended in GN inoculating fluid. Each
well of the Microplate was inoculated with 150 μl of bacterial suspension and the
plate was incubated in 28°C for 24 hr. Subsequently the plate was read by a microplate
reader and the read-outs were analyzed with MicroLog3 4.20 software.
Growth Experiments
If not stated differently, cells were grown on agar plates overnight in 30°C.
Transposon Mutagenesis
The mutants of
P. putida
were created using an
in vitro
transposition system (Epicentre
Technologies, Madison, Wisconsin, USA) [84]. This system bases on a hyper-reactive
Tn5-transposase and Tn5-Transposome that, in the absence of magnesium ions, builds
a stable synaptic complex, which can be transmitted into the cell via electroporation.
