Glycine Assay Protocols

Three glycine assays were performed in order to better characterize the glyA Keio Knockout Strain (JW2535-1). These included an assay to determine the minimal amount of glycine required for survival of the auxotroph in minimal media which was used in previous Petrobrick assays. Additionally, the functionality of the Petrobrick was evaluated in this strain to see if similar concentrations of hydrocarbons could be produced as in WT strains of E. coli. In order to evaluate if this strain could be used in conjunction with our kill switch mechanisms, we used these systems in conjunction.

Media Optimization for the Glycine Auxotroph

• The glyA auxotroph was grown in LB media overnight at 37^oC with shaking.
• Cells were washed in M9 minimal media two times and spun in between at 4,000 RPM at 4^oC for 10 min. each time.
• Finally, washed cells were subcultured into 5 mL of M9 minimal media supplemented with 1% (w/v) glucose and 50 ug/mL Kanamycin.
• Cells were grown at 37^oC with shaking for 12 and 24 hours and OD₆₀₀ measurements were used to quantitate growth.

Glycine Auxotrophy Effect on Petrobrick Output

The effect of the auxotroph on the output of the Petrobrick was determined as discussed in the flux balance analysis assay using 50 mM glycine. Click here to view this protocol.