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Riboswitch

Ribozyme

Bidirectional pTeT Controlled Kill Switch Growth Assay

The kill switch was assayed by measuring the OD of cultures subjected to varying levels of the inducer. While not intended to be used in final probiotic, the tetracycline analogue, anhydrotetracycline (ATC), has been shown to have a lower antibiotic activity and affinity for TetR and was thus chosen for use in our experiments. By comparing the growth of cells transformed with a plasmid lacking the promoter and cells with a plasmid lacking the killswitch and the promoter, we showed that cell growth was not affected by the kill switch.

Cell growth was unaffected by leaking of the promoter in cells with the bidirectional kill switch construct, showing that our device will not restrict the ability for the chassis to colonise the target region of the gut.

Our plate reader assay shows that the bidirectional promoter/kill switch construct induces cell lysis in response to concentrations of ATC equal to or above 1nM. Higher concentrations increase the rate of cell lysis up to concentrations of 20nM. Above this concentration, the promoter is at maximum activity.

Cell growth for negative controls, while higher than cells with the kill switch construct, was reduced by high ATC concentrations. This shows that while ATC has less antibiotic activity than tetracycline, an analogue with lower antibiotic activity or increased binding affinity for TetR is required for a highly selective and effective inducible kill switch.

Unidirectional pTet Controlled Kill Switch Growth Assay

Bidirectional Promoter Fluorescence Assay

In addition to characterising the kill switch under the control of the bidirectional Tet promoter, we decided that characterising the strength of the promoter by looking at the production of sfGFP would provide critical information for future experiments. The obtained parameters would allow future models to accurately predict expression of a new part in response to known levels of ATC.

To accurately measure the activity of the promoter, two distinct constructs were used. the negative control construct contains an RFP gene under the control of the bidirectional promoter in one direction and TetR in the other. The expression vector we tested in the assay was identical to the negative control however RFP was replaced with sfGFP, the fluorescence of which was measured.

By finding the concentration at which the fluorescence is equal to in the absence of inducer, we can determine that the promoter is insensitive to concentrations below [sdfdfsdfs]nM.

Our data also was used to determine the leakiness of the promoter. Basal gene expression was obtained by measuring fluorescence in the absence of ATC relative to the negative control. This comparison was critical for isolating that the only contributing factor was the promoter inhibited by TetR.