Team:William and Mary/Measurement
We performed a number of rigorous and thorough measurements, on each component of the circuit control toolbox. We measured the effect of the insulating ribozyme RiboJ on relative fluorescence, and confirmed that it does indeed insulate proteins from genetic context. (Figure 1). We also measured RiboJ’s effect on absolute expression, and discovered that it increases absolute expression (Figure 2). We created a RiboJ promoter library containing the Anderson Library of Promoters as well as additional inducible promoters, this library will form the basis for an ongoing rigorous characterization of the effect of RiboJ on absolute fluorescence.
We also characterized the community collection of RBSs under the inducible promoter pLacO-1 (Figure 3). While our characterization was mostly in line with existing values, it highlighted the need to characterize over a range of conditions rather than at just one level of expression. Since our characterization was insulated using RiboJ, it should be applicable in all genetic contexts, even though past characterizations may not be. Additionally, we collaborated with UPitt and Alverno to measure our RBS characterization parts in cell free systems (Figure 4 and 5). Characterization was also performed on the Interlab parts using FACS.
Finally, we performed the first iGEM characterization of two new parts, which allow for orthogonal control over the output of a given arbitrary circuit. We replicated and characterized both the original and our improved synthetic enhancer on the Biobrick Backbone, which allows for multistate output beyond a normal transfer function (Figures 6-7). We also performed characterization of the effect of molecular titration on transfer functions by using a TetO array to cause a leftward shift (Figure 8). A model was made based on literature values, and our characterization matched our models prediction (Figure 9).
Figure 1: RiboJ acts to homogenize translational efficiency between promoters by cleaving the 5’ region upstream from
RiboJ. -35 and -10 boxes are shown in bold, and transcription factor binding sites are underlined. Note how there is
an operator sequence located within the transcribed region of the pTac promoter (BBa_K864400), and that pSal and pBad
include transcribed regions. These untranslated regions can change translational efficiency of the downstream protein,
leading to inconsistent expression between different combinations of promoters and coding sequences. Figure modified
from figure S1 of Lou et al. 2012.
1. C. Lou, B. Stanton, Y.-J. Chen, B. Munsky, C. A. Voigt, Ribozyme-based insulator parts buffer synthetic circuits from
genetic context. Nat. Biotechnol. 30, 1137 (2012). doi:10.1038/nbt.2401 pmid:23034349
Measurement
References
