Team:Oxford/results
Results
Riboswitch Fluorescence Assay
The riboswitch parts were characterised by inserting a unidirectional Tet promoter before the upstream folding region and after it. The cells transformed with these constructs were then grown in LB at 37oC for 3 hours. This would allow us to assess this importance of this region in the sensitivity to the binding of adenine. We decided to use a plate reader assay which would measure the fluorescence of eCFP at 480nm. Adenine has a solubility in water at pH 7 of 7.4uM which is insufficient to accurately measure the mass of solute for a stock solution.
In preliminary experiments, adenine was dissolved in 100% DMSO to a stock solution of 15uM. The DMSO concentration was shown to be high enough to inhibit cell growth. We believe that this will have altered the sensitivity of the riboswitch and invalidated our results.
To overcome this issue, we changed our inducer from adenine to adenine sulfate dihydrate which is soluble in water to 15uM. We repeated the assay using this new stock.
Ribozyme Fluoresence Assay
Although we could not assemble a functional ribozyme in the shipping vector, we had expression vectors which used sRNA to repress sfGFP, controlled by a common pTet. Using these plasmids, we could use to validate our decision to include it in the design. To confirm the neccesity of the ribozyme in activating the sRNA, we performed a plate reader assay for sfGFP by measuring the fluoresence at 520nm in response to 10nM ATC in a range of constructs with fluorescent proteins under the control of a bidrectional pTet.
As shown in the results of the assay, the ribozyme is critical in the functioning of the sRNA. The construct with the human hammerhead ribozyme is the only construct in which the sRNA can repress translation of sfGFP mRNA.
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 at 520nm.
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.
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
The assay for the unidirectional kill switch was performed using the same protocol as the bidirectional kill switch. Although sequencing showed the SDM occured, we believe base deletions during the PCR may have resulted in a loss of function mutation in the promoter. The graph below shows that there is no significant lysis of cells as a function of ATC.| Index | Reference |
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| A | https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/a8626pis.pdf |