Team:Oxford/Protein Based Experiments

Future Protein-Based System Experiments

Introduction

In our project, the central sfGFP-dark quencher part has been shown to have repressed fluorescence which is relieved on cleavage by TEV, and this fluorescence is unaffected by the presence of the TorA leader sequence, spycatcher and His tag. This represents the first design built test cycle, proving the concept of relieved repression at this point, and as will be seen, this will be used throughout our suggestions for future experiments. We have also designed the parts that would be used to target proteins to the membranes, and will explain the future experiments to test these.

DBT Cycle 1 - Membrane Insertion and OMV Packing Efficiency

Next DBT- design, build and test membrane insertion efficiency, and the packing efficiency into the OMV. Express the parts BBa_K2450401 and BBa_K2450451, which have SpyTag at the N terminal and in the middle of the protein respectively. Localisation of the fluorescence of these parts should be seen in the outer membrane, which can be performed by microscopy. Once it is confirmed that both these parts can be expressed in the membrane, the sfGFP should be removed by PCR, and subsequent experiments carried out in the absence of the fluorophore to avoid interference with other fluorescence experiments. The SpyCatcher-sfGFP part (with the dark quencher removed during PCR) should then be expressed in the same E. coli strains. These will then be transported into the OMVs which can be extracted by ultracentrifugation using the protocol shown here. The fluorescence of these OMVs can be compared, and the highest fluorescence will be the most efficient at targeting protein to the OMVs. This targeting protein can then be used in all subsequent experiments.

DBT Cycle 2 - OMV Lysis Method

After identifying the most efficient targeting protein, it is then possible to start assaying OMV lysis. We have proposed using a detergent, such TritonX or some other ‘soft’ detergent. The detergent should be able to be added in a powdered form, should not denature proteins, and should be cheap. It may be that future teams would like to test other methods, such as increase in temperature or sonication, which are tested in the same way. In assaying the OMVs, TEV protease should be added first and left, to ensure no fluorescence change occurs and that the OMVs are intact. If this is not the case, then the TEV protease + OMV mixture should be left until the fluorescence plateaus, and this taken as the new zero. The method being assayed should then be used (e.g. adding the detergent) and the fluorescence should increase as the OMVs are lysed and the TEV protease gains access to the SpyCatcher-sfGFP-TEV cleavage site-dark quencher. This assay has the advantage that if the lysis technique begins to lyse proteins, this will be seen as a decrease in fluorescence, and can be observed by its self by lysing the OMVs in the absence of TEV protease.

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