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gRNA vector


We created a gRNA expression vector gRNA-pSB1C3 (BBa_K2515002). We successfully cloned our gRNAs into it and confirmed their functionality via transformatin in E. coli competent cells with Cas9. As expected no colonies were obtained.



Then we cloned a couple of gRNAs for the ftsZ gene. Upon arabinose induction we observed a trend to elongated cellular shape (see the picture below - left is ftsZ gRNA, right - control). This was a positive trend no matter that we expected a stronger phenotype based on literature data.



This observation was supported by the fact that ftsZ gRNA containing cultures develop much more intensive red color (see the picture below - 3 left tubes are ftsZ gRNAs, right - control). The more intense color can be explained with inhibition of the cellular division to a given extent and redirection of the metabolic flux to side processes like chromoprotein production.



Mutator Strain



We tested our mutator strain via plating cultures with gRNA arrays that suppress genes with an important role in different reparation and/or replication fidelity systems. As a readout we used the number of rifampicin resistant colonies. Unfortunately our results so far are inconclusive due to the small experiment number. We hope that this will change until our presentation on the Giant Jamboree.