Team:KU Leuven/Design

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Phased approach

Here we describe the general overview of our project in terms of design on the wet lab front. We effectively split our project into three main phases to reach our final goal of adapting EvolvR in planta:

Phase 1: Testing EvolvR in bacteria
In order for us to introduce EvolvR into a new biological system, we sought to confirm the efficiency and functionality of EvolvR in bacteria as previously described [1]:

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Figure 1. A nonsense mutation is first introduced in a superfolder green fluorescent protein (sfGFP) gene via site-directed mutagenesis. E. coli cells containing ΔsfGFP are then transformed with a pEvolvR plasmid with a gRNA targeting the nonsense mutation in ΔsfGFP. If EvolvR is functional, it would revert the mutation back to wildtype and restore sfGFP back to wildtype.

Phase 2: Testing EvolvR in a cell-free system made from plant cell lysate
Before testing EvolvR directly in plant cells, we wanted to validate its functionality in a eukaryotic cell environment by testing it in a cell-free system made from Tobacco BY-2 cell lysate:

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Figure 2. We would use the same mutant GFP gene as a reporterto confirm the functionality and efficiency of EvolvR. Sequencing of the GFP gene after expression of EvolvR in the system will help us assess the targeted mutation rate of EvolvR in this system.

Phase 3: Incorporation of a functional EvolvR in BY-2 cells
Our ultimate goal being to validate that EvolvR can function as expected in BY-2 plant cells, we established a rapid method for a continuous directed evolution experiment with BY-2 cells:

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Figure 3. Agrobacterium tumefaciens transformed with a plasmid containing EvolvR with a gRNA that targets the BASTA herbicide susceptibility gene are used to transform plant cells. We would then grow the transformed cell culture in the presence of BASTA therefore selecting for cells that acquired resistance via mutations introduced by EvolvR.

Additional experimental design

Molecular ruler experiment
To measure the editing window of EvolvR, we designed ten evenly distributed protospacers throughout the GFP gene:

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Figure 4. Ten evenly distributed protospacers have been designed throughout the sfGFP gene. By running a continuous directed evolution experiment with each protospacer in parallel, we can measure EvolvR editing efficiency and its optimal editing window.

Riboswitch experiment
Because we do not have an inducible system in plants for EvolvR, we believed that evolving a riboswitch could be an avenue for us to implement such a system in a plant vector.

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Figure 5. We chose to use a theophylline-responsive riboswitch already reported in the iGEM registry to improve upon its characteristics, such as dynamic range and leakage, using our EvolvR system in E. coli for continuous directed evolution. A selection process was thoroughly worked out using the dual-selection marker of a TetA gene fused to sfGFP.

Establishment of a CMC suspension culture
Beyond the BY-2 suspension culture, we believe that establishing a suspension culture from CMCs, plant stem cells, will be beneficial for the implementation of EvolvR in plant biotechnology research.

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Figure 6. Our vision for an EvolvR experiment in CMCs. Agrobacterium tumefaciens transformed with a plasmid containing EvolvR with a gRNA that targets a gene of interest are used to transform CMCs. The transformed cells are plated on solid medium and transferred into a bioreactor to create a transgenic EvolvR CMC line that will be grown under selective pressure to evolve the target of interest.


[1] Halperin, S. O., Tou, C. J., Wong, E. B., Modavi, C., Schaffer, D. V., & Dueber, J. E. (2018). CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window. Nature, 560(7717), 248–252.