Team:Edinburgh OG/Results/Lambda

PhagED: a molecular toolkit to re-sensitise ESKAPE pathogens

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Production of Lambda phage with SaCas9/FnCpf1

To introduce the CRISPR systems into &lambda, both SaCas9 and FnCpf1 were separately inserted into the P1 phagemid and then digested/ligated with the &lambda using BsiWI and AvrII. Electroporation was used to introduce the engineered λ (SaCas9-λ and FnCpf1-λ) into our testing E. coli platform (MoClo). The production of engineered phages required a long period during which the electroporation was tested and optimised. The electroporation test performed in E. coli with the pGEX-4T-1 plasmid was successful and we moved to the optimisation step. The technique was optimised by separately varying the voltage, incubation period, exposure time, media but none of them resulted in an optimisation of the technique. During the electroporation optimisation with λ (not engineered), a single plaque was found in different replicates/optimisation steps (Figure 1) suggesting the inconsistency of the technique. When performing the electroporation with both the engineered λ, no viable recombinant phages were obtained and this part of the project could not be completed. The overall failure of the electroporation was probably due to the presence of active restriction systems in E. coli JM1 which destroyed the λ genome upon infection. Moreover, λ purchased was produced from a different strain of E. coli (GM33). Phages are sensible to the change of host and different chassis can be incompatible. Finally, the JM1 strain carries several mutations which make this strain fragile thus hindering the capability to uptake DNA and produce exogenous proteins.

Figure 1: Electroporation optimisation: only one plaque (red circle) was found in two conditions (1.5 kV - plate 1- and 1.75 kV - plate 2)

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P1 Lambda T4 T7 MoClo Model