Difference between revisions of "Team:William and Mary/RBS"

One of the more familiar ways of tuning a biological circuit is to change the strength of a Ribosome binding site (RBS). Changing the strength of an RBS allows for the alteration of the amplitude of a given transfer function. While this is a familiar concept to most iGEM teams, it takes on a special importance in the context of the Circuit Control Toolbox. This is because while transfer functions are preserved when shifted, the amplitude of those transfer functions is oftentimes reduced due to increased metabolic load. So we characterized a library of variable strength RBSs for use in amplitude tuning in the Circuit Control Toolbox.

While it would have been possible to construct brand new RBSs for use in the Circuit Control Toolbox, we thought that it would be more useful to characterize existing commonly used RBSs on the registry. So we chose to characterize the Community Collection, which is made up of 8 variable strength RBSs, and is commonly used by most iGEM teams.

For our characterization we chose to emulate the methods of Lou et al. 2012 (“Ribozyme-based insulator parts buffer synthetic circuits from genetic context”) and used a super folder Green Fluorescent Protein (sfGFP) with RiboJ under the control of pLacO1 (Bba_K2066014). The use of the IPTG inducible promoter pLacO1 allowed us to characterize the functions over an induction curve, and the use of RiboJ, a self cleaving ribozyme from Lou et al. 2012 prevented the 5’ untranslated region of the promoter from effecting the translational efficiency of the mRNA transcript (Figure 1). That means that unlike past iGEM characterization, our results are generalizable beyond the promoter pLacO1. (See the RiboJ section for more details)

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.

3A

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.

4A

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.

We held a science workshop targeted at at local children and their parents which allowed them to get some hands-on experience with the exciting world of modern biology. We had more than 70 people in attendance and together we extracted DNA from strawberries, learned about synthetic biology and the benefit of standard biological parts, and discussed our questions and concerns with GMO food.

We also held a forum on the topic of Genome Editing targeted at the general public. At the forum, we briefly discussed the implications of the CRISPR/Cas9 genome editing tool and explored various ways that it could be applied to real world problems. Among the topics discussed were: stabilizing the Honeybee population by increasing expression of ‘hygiene’ genes, engineering yeast for efficient biofuel production, and using CRISPR to decrease human susceptibility to HIV infection. We had over 30 participants for the forum and we received great feedback from our attendees!