Part:BBa_K2066047:Experience

Characterizations of BBa_K2066047

First, all RBS library parts were sequenced confirmed using Sanger sequencing.

In order to functionally characterize our RBS library BL21 E. coli were transformed with one of our standard characterization devices (BBa_K2066035, BBa_K2066036, BBa_K2066044-K2066049). All constructs were characterized on the pSB1C3 high-copy backbone. Cells were then selected for with antibiotics and individual colonies were grown overnight in M9 minimal media.

Cells were collected at log phase (OD ~ 0.4-0.6) for analysis. Culture was diluted 1:10 in 1x PBS and run on a BioRad S3e cell sorter. For each initialization of the cell sorter we also ran Spherotech RCP-30-5A Rainbow Calibration Particles to convert arbitrary fluorescence units into absolute Molecules of Equivalent Fluorescence (MEFL).

Raw FCS files were analyzed using FlowCal from the Tabor lab to automatically gate the cell populations based on FSC and SSC (Castillo-Hair et al., 2016).

The graph below shows the results of our characterization efforts. Solid lines are the mean of three biological replicates, and shading represents +/- standard error of the mean.

References: Castillo-Hair, Sebastian M., et al. "FlowCal: A user-friendly, open source software tool for automatically converting flow cytometry data from arbitrary to calibrated units." ACS synthetic biology (2016).

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