Team:Hamburg/Design
We designed a novel system to allow transformation of multiple plasmids with a single antibiotic resistance. Our system uses a toehold switch based AND logic. For more information visit our project description. The toehold switch implementing the AND logic and the corresponding triggers were adopted directly from Green et al. The we used the gate “ACTS_TypeII_N2 gate” [2] and the corresponding triggers. We chose this gate due to its low relative error.
We designed three plasmids as backbones for our different selection functions. We chose strong constitutive promoters from the Anderson Family for expression of the gate-resistance construct and the triggers. To make sure that the expression of the resistance gene is sufficiently high, even if the gate has a repressive effect. For the triggers we chose a slightly stronger promoter than for the gate-resistance construct to make sure that the gate is always sufficiently saturated with triggers when all plasmids are present.
The plasmid with the gate-resistance construct consisted of the promoter BBa_J23102, followed by the AND logic gate with chloramphenicol acetyl transferase as our output gene. We chose the strong terminator BBa_B1002. This is our composite part BBa_K2970006. The plasmids with the triggers consisted of the promoter BBa_J23100 followed by the triggers. We chose the strong terminator BBa_B1002. These are our composite parts BBa_K2970003 for trigger one and BBa_K2970004 for trigger two. As backbones we wanted our plasmids to only carry the sequences for their respective function in our selection system but this introduced a problem in handling our plasmids. Since none of the plasmids were viable on their own it would have been very difficult to handle them. For this reason we cloned the respective sequences into a pSB1A3 backbone for easy amplification. We modified this backbone with BsmBI cut sites on both sides of the ampicillin resistance so we could remove it before testing our own selection system. To test the functionality of our selection system we designed simple qualitative transformation experiments. We transformed bacteria with all three plasmids of our selection system on chloramphenicol plates. If these transformations are unsuccessful our selection system doesn’t work. If they are successful we still need to confirm that transformations with a subset of our plasmids are unsuccessful. Otherwise complete transformation of all three plasmids is not guaranteed. Our transformation experiments showed successful transformations for any sample containing the gate-resistance construct. We believe that this observation is due to the gate leaking. The selection system designed in this project expresses a lower number of resistance proteins when compared to a typical selection system in which every plasmid type carries a different resistance gene. We want to show that this difference in number of expressed proteins also increases the bacterial growth rate. We will record growth curves for our selection system and also for a regular selection system by transforming bacteria with the empty iGEM backbones pSB1A3, pSB1C3 and pSB1K3. As our selection system depends on the relative strength of the promoters expressing the gate-resistance construct and the triggers we needed to confirm the relative and absolute strength of our promoters (BBa_J23100 and BBa_J23102).
To determine the promoter strength we cloned the promoters with the GFP part BBa_E0240 as a reporter into pSB1C3 backbones. We then transformed E. coli with this construct and measured the expression of GFP with FACS and plate reader images. Design
Design of triggers and gate
We modified the gate sequence by repeating the linker sequence. We wanted to increase the gates repression rate by adding additional secondary structures through repeated sequences.Expression of selection functions
Amplification of our plasmids
Experimental design
Revision: Comparing the gate plasmid with and without triggers
The gate has a high ON/OFF rate so the expression of the resistance is significantly higher when the triggers are expressed alongside the gate. We examined the impact of higher concentrations of antibiotics on the viability of transformants with the gate and with or without the triggers. For certain concentrations of antibiotics bacteria with just the gate will stop being viable while bacteria with the gate and both triggers will remain viable due to the higher expression of the resistance gene.Impact of plasmid type on growth rate
Promoter characterization
Sources
logic gates: [1] Green, A. A., Kim, J., Ma, D., Silver, P. A., Collins, J. J., & Yin, P. (2017). Complex cellular logic computation using ribocomputing devices. Nature. https://doi.org/10.1038/nature23271
toehold switches: [2] Green, A. A., Silver, P. A., Collins, J. J., & Yin, P. (2014). Toehold switches: De-novo-designed regulators of gene expression. Cell, 159(4), 925–939. https://doi.org/10.1016/j.cell.2014.10.002
Parts: http://parts.igem.org/Part:BBa_E0240, http://parts.igem.org/Part:BBa_J23100, http://parts.igem.org/Part:BBa_B1002