We also use the reaction rates from Equation 1 to write down a master equation, which has been simulated using the Gillespie algorithm to obtain the results in Figures 2 and 4. We have performed Linear Noise Approximation analysis to prove the robustness of our results, as described below.
Novel mono N-alkyl quaternary ammonium salts (3a-f) were prepared using the Menschutkin reaction from the cage adamanzane type aminal 1,3,6,8-tetraazatricyclo[4.3.1.13,8]undecane (TATU) and alkyl iodides, such as methyl, ethyl, propyl, butyl, pentyl and hexyl iodide (2a-f), in dry acetonitrile at room temperature.
Genotyping was carried out using the SNaPshot reaction from Applied Biosystems, which is a modified fluorescent single base pair extension procedure.
For the remaining essential gene pairs, the reactions associated with both genes are used in parallel in the wild type, a case of metabolic plasticity [ 61], or the reactions from only one of the genes is used while the reaction from the other member of the pair can act as a backup, a case of metabolic redundancy [ 61].
After identification, we used the LR reaction from the Invitrogen Gateway system to transfer the prey coding sequence from the yeast-destination vector to the entry vector.
The metabolic reconstruction algorithm was implemented as a Perl script, using the Chemistry::Reaction module from the PerlMol collection of Perl modules for computational chemistry (Tubert-Brohman, 2004).
In order to measure the parameters of nucleotide binding prior to the phosphate transfer step, ATPγS, an ATP analogue resistant to hydrolysis was used to prevent the reaction from proceeding to completion which otherwise would result in a large heat change that will mask the heat change due to binding.
Serotyping was conducted by using the quellung reaction with reagents from Statens Serum Institut (Copenhagen, Denmark) (18).
The capsular serotype of all isolates was determined by using the Quellung reaction with antisera from the Statens Serum Institut (Copenhagen, Denmark).
