Team:UT-Knoxville/Methods

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Enzyme Characterization Testing

To begin characterization, BL21 DE3 + pXYLAMB (BBa_K1966003) was grown until OD600 > 2.0 in 50mL LB media + antibiotic. Cultures were then centrifuged for 20 minutes and resuspended in fresh LB at OD600 = 10.0. Enzyme production was induced with 2.5mM IPTG after resuspension and fresh antibiotic was added. 2.5mL of high cell density culture was loaded into 25mL culture tubes along with 5 mM of the substrate of interest and tubes were sealed to prevent evaporation of substrates and products. Cells were incubated with shaking at 37 &degC for 8 hours.

After incubation, sealed culture tubes were moved to an ice bath for 1 hour to condense volatile chemicals. When condensation was complete, products were extracted from culture and analyzed by GC-MS.

Gasoline and BTX Simulation Testing

Cells for these tests were prepared in a similar way to the enzyme characterization mentioned above. For gasoline testing, gasoline was added at 0.5% v/v to 2.5 mL high cell density cultures. For BTX simulation, toluene and o-, m-, and p-xylene were added at a total concentration of 5Mm. Sample extraction method remained the same as in basic substrate characterization.

GC-MS Extraction

Extraction solvent of hexane+25mg/L internal standard (isoamyl acetate) was prepared in bulk and used for all extractions. 500uL culture was added to equal volume hexane+IS and mixed well. Cells in this mixture were lysed via 5 minutes of beat beating with 60uL 6N HCl. Samples were placed in an ice bath for 5 minutes after bead beating and centrifuged for 10 minutes at 15,000 RPM. The separated organic layer was analyzed via GC-MS.

Indigo Production and Extraction

Cells were centrifuged at 15,000 RPM for 20 minutes and supernatant poured off. Cell pellets were resuspended in a 2:1 v/v chloroform:methanol mixture and vortexed for 1 minute. Water was added at equal volume to the 2:1 chloroform:methanol and mixture was vortexed for 1 minute. This mixture was centrifuged at 15,000 RPM for 10 minutes and aqueous layer was pipetted away. The remaining solution of indigo in chloroform was left in a chemical fume hood overnight to allow solvent to evaporate, leaving behind indigo crystals.

Enhanced indigo production culturing conditions were developed. Cells were grown in LB for 8-12 hours until stationary phase was reached and resuspended in fresh LB at OD600 = 2. IPTG was added at 0.5mM and indole was added at 25mM. Cells were grown for 24 hours and indigo was collected. It is recommended to use large baffled culture flasks for maximum oxygen transfer (needed for reaction of indoxyl to indigo).

Pu Promoter Characterization

Cells were grown to stationary phase in LB (8-12 hours) and resuspended to OD600 = 1 for characterization. IPTG was added at 0.5mM with appropriate antibiotic. Substrates of interest were added at a concentration of 5mM each and fluorescence was measured via plate reader hourly.