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<h1>Results </br>(Protein-Based System)</h1> | <h1>Results </br>(Protein-Based System)</h1> | ||
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<h2> Introduction </h2> </br> | <h2> Introduction </h2> </br> | ||
<p>In order to demonstrate the feasibility of our Protein-based system for detecting Cruzipain, we designed and cloned three parts into pSB1C3:</br> | <p>In order to demonstrate the feasibility of our Protein-based system for detecting Cruzipain, we designed and cloned three parts into pSB1C3:</br> |
Revision as of 13:27, 1 November 2017
Results (Protein-Based System)
Introduction
In order to demonstrate the feasibility of our Protein-based system for detecting Cruzipain, we designed and cloned three parts into pSB1C3:
- SpyTag-OmpA-sfGFP-His
- OmpA-SpyTag-sfGFP-His
- TorA Leader-SpyCatcher-sfGFP-TEV cleavable linker-Reach quencher-His
From our modelling of this system we had calculated that we needed…. Blah blah blah Arthur please help.
Shipping Vector Cloning
A more in-depth description of this stage of our project can be found on the Shipping Vector Cloning results page. We cloned the three parts above into the shipping vector, however we had additionally designed 2 extra parts, two halves of a split TEV protease. These contained illegal restriction sites, and therefore couldn’t be added to the registry. We attempted quick-change PCR and overlap extension PCR for site-directed mutagenesis, and the two protocols are on our page, however these were unsuccessful. We therefore could not submit these parts.
Expression Vector Cloning
In order for full characterisation of both parts to take place we needed to express both of them in the same system, so we designed an experiment where one part with OmpA-SpyTag and one with SpyCatcher-GFP-quencher would be present on different plasmids in the same cell. We would clone the OmpA-SpyTag part into the pQE-60 vector, and the SpyCatcher-GFP-Quencher part into pBAD-33. These have compatible origins of replication, differing antibiotic resistances, and are induced by two different and easily-obtainable inducers, IPTG and arabinose respectively. We designed primers for three purposes:
- To amplify the parts from the shipping vector constructs with restriction sites that allowed for cloning into the expression vectors. These were NcoI and BamHI for pBAD-33 and XbaI and PstI for pQE-60. These enzyme combinations ensured that the start codon of the part was the optimal distance from the RBS in the plasmid for efficient expression.
- To amplify a ‘noF’ version of the OmpA-SpyTag part which would remove the GFP fluorophore and therefore fluorescence would only be detected in the OMV fraction if the SpyCatcher-sfGFP-Quencher part was localised. In hindsight a better design may have been to have used a different fluorophore to assay both at once. This could even have been a CFP in order to use FRET to see if the two parts were close to one another.
- To amplify a ‘noQuencher’ version of the SpyCatcher-sfGFP-Quencher part, in order to determine the effectiveness of quenching in our system.
SpyCatcher-GFP-Quencher Characterisation
Inital Microscopy to Show Fluorescence
The first experiments we ran were to show that sfGFP was produced, and that the quencher worked to reduce fluorescence. Initial microscopy showed that when the quencher was removed from the part the cells did exhibit fluorescence. We were unable, however, to see whether the TorA leader sequence had worked as planned, with the part, and therefore the fluorescence, transferring to the periplasm, which would have manifested as a ‘halo’ around the cell.Plate Reader Assay of Quencher
We then ran a plate reader assay what were the conditions of the assay , how did we get the graph? comparing the fluorescence of the quenched and unquenched part, again with an empty pBAD plasmid as a negative control. Induction with arabinose to produce the part caused an almost 10-fold increase in fluorescence in the noQuencher version of the part, with the levels of fluorescence in the quenched part not differing significantly from the control, effectively showing a quenching efficiency of 100% under these conditions.In vitro Cleavage Assay
We then looked to characterise relief of the quenching by cleavage with TEV protease. We would combine purified sfGFP+Quencher and TEV protease in a plate and let it run in the plate reader, to see relief of quenching. Originally we attempted to do this in vitro, by purifying the part and then adding purified TEV protease. We were helped greatly in this regard by Associate Prof. Maike Bublitz, from the Biochemistry department, who provided us with purified TEV protease and guided us on how to purify the protein using a nickel column, which was kindly lent to us by Prof. Matt Higgins, also from the department. It appeared from our SDS-PAGE gel, however, as if we may have used too strong a wash for our purification, and washed our protein from the column before we got to our elution fractions. We tried some of the fractions in the plate reader to be sure, however there was no increase in GFP over time.In vivo Cleavage Assay
Summary and Conclusions
Our Results
- We cloned three parts into the pSB1C3 shipping vector
- We cloned two versions of one of these parts into pBAD-33, our expression vector of choice
- We showed that the sfGFP was produced
- We determined that the quenching peptide was effective at quenching fluorescence
- We showed that this quenching could be relieved by cleavage by the TEV protease
Improving Characterisation of an Existing Part
The Aachen 2014 iGEM team from which we took the GFP-TEV cleavage-Reach2 section of our part showed in their system that the quencher worked and that it could be relieved by cleavage by the TEV protease. We have added to this characterisation by comprehensively showing that the same effects occur even with:
- The addition of a his-tag onto the quenching peptide
- The addition of upstream leader sequences (TorA and SpyTag)
- The substitution of GFP for sfGFP