Part:BBa_K2066031:Experience

All Ribozyme constructs were sequence confirmed using Sanger sequencing.

In order to functionally characterize our ribozyme parts BL21 E. coli were co-transformed with one of our RiboJ characterization devices (BBa_K2066014, BBa_K2066015, BBa_K2066024-BBa_K2066027, BBa_K2066030, BBa_K2066031) on pSB1C3 and the constitutive Lac repressor (BBa_K2066016) on pSB3K3.

Cells were then selected for with antibiotics and individual colonies were grown overnight in LB media.Cells were diluted 1:100 into fresh LB and growth to an OD of ~ 0.4 - 0.6 before induction with IPTG. After addition of IPTG cells were grown for six hours to allow for induction. 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 for . Solid lines are the mean of three biological replicates, and shading represents +/- standard error of the mean.

pTac does not require RiboJ to have a collapsed transfer function. This is in contrast to our results with pLACO-1 where the inclusion of RiboJ was a necessity. In order to allow future teams to use any promoter with out Circuit Control Toolbox we therefore suggest using RiboJ on all promoters if this characterization has not been performed.

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