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 1A
Figure 1B
Figure 1A and B: RiboJ insulates transfer functions from genetic context, collapsing the relative transfer functions of two different reporters.
Figure 2A
Figure 2B
Figure 2A and B: RiboJ has divergent effects on absolute expression on different promoters. RiboJ decreases absolute fluorescence under the promoter pLacO-1(A), but increases it under pTac (B).
Figure 3: Relative sfGFP characterization of the Community Collection of RBSs with RiboJ using the inducible promter pLacO-1.
Figure 4: Characterization (blinded) of our RBS library by University of Pittsburgh in their cell free system S30.
Figure 5: Characterization (blinded) of our RBS library by Team Alverno in their cell free system TX-TL.
Figure 6: Characterization of improved synthetic enhancer in Biobrick Backbone.
Figure 7: Comparison of characterization of original synthetic enhancer from Amit et al. 2012 and improved synthetic enhancer on Biobrick Backbone.
Figure 8: Effect of repeat TetO array on the induction curve of pTet GFP
RBS B0029 1 RBS B0029 2 RBS B0029 3 RBS B0030 1 RBS B0030 2 RBS B0030 3 RBS B0031 1 RBS B0031 2 RBS B0031 3 RBS B0032 1 RBS B0032 1 Email igem@wm.edu if you want to see the rest of our more than 200 FACS plots!
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
FACS Plots
References
