Team:UNILausanne/Measurement

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Measurements

Relevance

Genetically modified organisms can pose a risk for the environment and can be a potential danger to human health. As this is a widespread concern in society, many iGEM teams decide to include a killswitch in their design. This component ensures that their GMO is only alive in its intended environment of use.

For our project, we designed and tested a killswitch system based on two well-known toxin-antitoxin systems (Figure 1). These are ccdb (BBa_K1318000) and ccda (BBa_K1075032), and miniColicin E2 (BBa_K1976048) and IM2 (BBa_K1976027). We tested the effect of differential expression of toxin and antitoxin by using orthogonally inducible promoters pTet (BBa_K3482012) and pTac (BBa_K3482011), which can be induced with aTc and IPTG, respectively.

Figure 1 Inducible toxin-antitoxin testing plasmids. a) pAND-MSC (BBa_K3482021) is the empty vector used as negative control. b) pKA1 codes for the ccdb toxin and ccda antitoxin (composed of BBa_K3482016 and BBa_K3482020). c) pKC1 codes for miniColicin E2 toxin and IM2 antitoxin (composed of BBa_K3482004 and BBa_K3482014) d) pKC3 codes for miniColicin E2 toxin and heat-inducible IM2 antitoxin (BBa_K3482017).

Measurement

We measured growth curves in parallel using a 96-well plate reader to determine the size effect and duration of the growth inhibition for a gradient of inducer concentrations spanning five orders of magnitude. With this extensive characterization, we showed which expression levels of toxin and antitoxin are required to obtain desired growth outcomes (Figure 2,3,4).

Figure 2: Dose-response growth curve of E. coli Nissle 1917 ΔclbA harboring kill switch plasmid pKA1 at 37°C. E. coli Nissle with pAND (red line), E. coli Nissle with pKA1 (purple line). The lines and shade represent the mean ± standard error. Each plot shows the absorbance at 600 nm measured every 10 min for a period of 10 hours at different aTc and IPTG concentrations.

Figure 3: Dose-response growth curve of E. coli Nissle 1917 ΔclbA harboring kill switch plasmid pKC1 at 37°C. E. coli Nissle with pAND (red line), E. coli Nissle with pKC1 (green line). The lines and shade represent the mean ± standard error. Each plot shows the absorbance at 600 nm measured every 10 min for a period of 10 hours at different aTc and IPTG concentrations.

Figure 4: Dose-response growth curve of E. coli Nissle 1917 ΔclbA harboring kill switch plasmids pKC1 and pKC3 at 37 and 25°C. E. coli Nissle with pAND (red line), E. coli Nissle with pKC1 (green line), and E. coli Nissle with pKC3 (blue line), at 37°C (dashed line) and 25°C (solid line). The lines and shade represent the mean ± standard error. Each plot shows the absorbance at 600 nm measured every 10 min for a period of 10 hours at different aTc and IPTG concentrations.

The observed pTet and pTac promoter induction can be matched to the corresponding GFP dose-response measurements for pTet and pTac, allowing the user to then choose a constitutive promoter of corresponding strength in, for example, the Anderson promoter catalog.

Achievement

With our measurements we therefore lay the foundations for a rapid and standardized implementation of killswitch solutions based on ccdb/ccda and miniColicinE2/IM2 for the iGEM community. Future teams will be able to implement similar kill switch mechanisms to their projects, as a way to reinforce biosecurity in their synthetic biology projects. We think it is important that teams star taking into consideration ways to reinforce containment measures to prevent accidents and unwanted propagations.