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.
Characterization was done on 07/26/16 using flow cytometry, which means that our data shows single cell resolution of the
impact of various RBSs. Characterization was done in BL21 E. coli, which is a standard protein expression strain. A high
copy plasmid (1C3) containing one of the pLacO1 RiboJ RBS variants (ex. Bba_K206636) was co-transformed with a low copy
plasmid (3K3) which contained a constitutively expressed LacI (Bba_K206616) to repress the promoter. Individual colonies
were picked from antibiotic selection plates, and colony PCRed to ensure the correct plasmids were contained. For each RBS
variant 3 colonies containing both plasmids were inoculated in M9 and incubated till midlog growth was reached. At that
point the cells were induced with various concentrations of IPTG until steady state was reached. Cells were then FACSed
and their fluorescence was reported in MEFL calculated using Spherotech calibration beads. (Figure 2).
Figure 2: Average (3 biological replicates) population level fluorescence of RBS library over different induction conditions.
Note the wide range of RBS strengths in the library, and that the strongest RBS B0035, is not actually the strongest until
induced at a concentration of 1000µM IPTG. This illustrates the importance of evaluating over a transfer function, not just
at an arbitrary steady state.
All of the individual RBS plots are available on our measurement
page, but here are a few examples of individual RBS measurements.
Figure 3A
Figure 3B
Figure 3A and 3B: Fluorescence measurements of replicate 1 of B0031 (BBa_K2066036) un-induced (A) and induced at [IPTG]
100µM (B). Note the relative crispness of peaks, indicating a nearly normal distribution of florescence. The majority
of all replicates are similarly unimodel in their distribution.
Figure 4A
Figure 4B
Figure 4A and 4B: Fluorescence measurements of replicate 3 of B0031 (BBa_K2066036) un-induced (A) and induced [IPTG] 100µM(B).
Note the bimodal distribution compared to figure. In this case induction occurs, but a subset of the population is maximally
induced at all induction conditions. All induction conditions of number 3 contained this distribution, while other replicates
of this biological circuit had a more normal fluorescence distribution, indicating that something unusual, likely the loss of
the repressor is going on in this replicate. Of note, this bimodal distribution would not be noticeable without the use of FACs
to observe at a single cell level.
While teams have measured RBSs in the past (Warsaw 2010), to our knowledge no team has ever measured the entire community
library over a variety of induction conditions. As illustrated by B0035’s variable relative strength at different induction
conditions, it is vital to perform characterization at different induction conditions rather than just constitutively.
However, our findings have been consistent with the measurements on the registry, and much like Warsaw 2010 we found that
B0030 was stronger than reported in previous measurements on the registry. Additionally, measurements on the registry by Kelly
and Rubin in 2007 indicate that B0035 is stronger than B0034. Given that their data was recorded from a strong constitutively
expressed promoter, their measurement makes sense in the context of our findings that B0035 is a stronger RBS only when expressed
at a high level.
While the community RBSs are designed for and most commonly used for expression in E. Coli, we thought that it would be
useful to characterize them in a variety of conditions. So as a collaboration we sent a blinded version of the library to
University of Pittsburgh’s iGEM team, who then proceeded to characterize the RBSs in the cell free system S30 (Figure 5).
We also sent a blinded library to the high school team Alverno, who characterized them using Richard Murray’s TX-TL cell free system
(Figure 6). In both cases, the majority of RBSs had relative expressions similar to our characterization. However, Team Alverno
characterized B0064 as having a significantly higher relative expression than was found in existing registry characterization as
well as our characterization. We talked to UPitt iGEM, and they noted that due to technical concerns they were not confident about
the validity of their first set of measurements (Figure 7). When we looked at the unblinded data from the first set of measurements,
we noted that they had a similarly high level of B0064 expression. Future experiments are needed to determine whether that is an
artifact of a common procedural error in cell free systems, or is representative of a novel result in a cell free system.
Figure 5: Unblinded graph of UPitt iGEM’s measurements of our RBS library in the S30 cell free system. Their data
is in line with both existing measurements, as well as our measurements.
Figure 6A: Unblinded graph of Team Alverno’s characterization of our RBS library in the TX-TL cell free system.
Figure 6B: Time course measurements of Team Alverno’s characterization of our RBS library in the TX-TL cell free system.
Our RBS characterization highlights the importance of rigorous characterization, especially in the context of our
Circuit Control Toolbox. To be able to use RBSs to tune the amplitude of any given transfer function, you need to be
sure that you can apply your characterization to a given situation. For example, using our RBS characterization it
becomes clear that it is important to consider both the system you are working in as well as the number of mRNA
transcripts created. Someone trying to use B0035 for high expression of a protein under the control of a weak
constitutive promoter needs to be aware that B0035 is only the strongest relative RBS when there are a large number
of copies of mRNA. Keeping this in mind, it is now possible to use our characterization to tune the amplitude and
relative max of various transfer functions. Using RiboJ to remove 5’ untranslated regions, and keeping in mind the
various limitations of any set of characterizations, our characterization of this will be of use in the Circuit Control
Toolbox, and for teams at large.
Figure 7: Team Pitt’s first series of measurements
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
RBS Tuning
References
