Difference between revisions of "Team:Oxford/results"
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<h2>Kill Switch</h2> | <h2>Kill Switch</h2> | ||
| − | <p class="lead">The kill switch was assayed by measuring the OD of cultures subjected to varying levels of the inducer | + | <p class="lead">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 inducer tetracycline analogue has been shown to have a lower antibiotic activity and affinity for TetR and . 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. <p>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. |
<p>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. | <p>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. | ||
<p>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. | <p>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. | ||
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<h2>Bidirectional Promoter</h2> | <h2>Bidirectional Promoter</h2> | ||
| − | <p class="lead">In addition to characterising the kill switch under the control of the bidirectional Tet promoter, we obtained.</p> | + | <p class="lead">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 production of a fluorescent protein 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.</p> |
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Revision as of 18:37, 17 October 2018
Results
Section 1
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Kill Switch
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 inducer tetracycline analogue has been shown to have a lower antibiotic activity and affinity for TetR and . 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.
Bidirectional Promoter
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 production of a fluorescent protein 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.