Flow cytometry analysis of P(3HB) production in <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i> from waste-media cultures.

<p><i>E</i>. <i>coli</i> MG1655 transformed with either empty vector [EV], native [N], constitutive [C] or hybrid [H] <i>phaCAB</i> constructs were cultured in 5 ml of waste-media for 36 h at 37°C. P(3HB) content was assessed via flow cytometry analysis of Nile Red staining. (<b>A</b>) Representative forward scatter (FSC) and side scatter (SSC) contour plots. (<b>B</b>) Representative histogram (FL-5). (<b>C</b>) Normalized fluorescence of Nile Red stained <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i>, from three independent experiments. Error bars, +/- the standard deviation. Student t-test, *P<0.05 and ***P <0.001.</p

P(3HB) production in <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i>.

<p><i>E</i>. <i>coli</i> MG1655 transformed with empty vector, native, constitutive or hybrid <i>phaCAB</i> constructs were cultured in 1 liter LB media, supplemented with 3% glucose (w/v) for 24 hours or 48 hours. P(3HB) was purified from these cultures and measured as <b>(A)</b> P(3HB) production (g/L) and <b>(B)</b> P(3HB) content (weight [wt.] % of cell dry weight [CDW]). Data represent the mean +/- the standard deviation of three independent experiments. Student t-test, *P<0.05, **P <0.01, ***P <0.001 and ****P <0.0001.</p

<i>phaCAB</i> pathway and constructs.

<p>(<b>A</b>) Schematic of poly-3-hydroxybutyrate (P(3HB)) production via the <i>phaCAB</i> operon pathway. (<b>B</b>) The constructs used in this study. Abbreviations: Pwt (wildtype promoter; green arrow), J23104 (Anderson constitutive promoter, BBa_J23104; red arrow), B0034 (ribosomal binding site, BBa_B0034; red half-circle), <i>phaC</i> (PHA synthase), <i>phaA</i> (3-ketothiolase) and <i>phaB</i> (acetoacetyl-CoA reductase). Green half-circles denote native ribosomal binding sites. Construct symbols are based on the Synthetic Biology Open Language Visual (SBOLv) v1.0.0 guidelines [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117202#pone.0117202.ref021" target="_blank">21</a>].</p

Simulated P(3HB) production in <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i>.

<p>In order to simulate P(3HB) production in <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i>, a P(3HB) synthesis model was constructed using the Simbiology toolbox of Matlab. Using this model the flux of several metabolites and species were simulated in order to identify aspects of the system that could be selectively tuned to increase the production of P(3HB). From these analyses, several novel <i>phaCAB</i> operons were designed. These data show the simulated P(3HB) production across several different <i>phaCAB</i> operon designs, where <i>phaCAB</i> expression is under the control of the indicated Anderson constitutive promoters.</p