Team:Oxford/Results Protein

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:

  1. SpyTag-OmpA-sfGFP-His
  2. OmpA-SpyTag-sfGFP-His
  3. 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:

  1. 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.
  2. 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.
  3. To amplify a ‘noQuencher’ version of the SpyCatcher-sfGFP-Quencher part, in order to determine the effectiveness of quenching in our system.


We successfully cloned the SpyCatcher-GFP-Quencher part into pBAD-33, however were unable to clone the OmpA-SpyTag part into pQE-60. This limited the experiments we were able to do with OMVs, however we were able to characterise the quencher, ensuring that if future teams can express the two parts together they will be able to use the finished system as an assay for protease presence. This can either be for the presence of protease which has been localised to OMVs or for the lysis of OMVs, by adding protease to the media they are in.

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


Following this setback, we instead decided to characterise the cleavage in vivo. A member of the Biochemistry department, Matteo Ferla, kindly gave us some TEV plasmid which was compatible with our part, and was IPTG-inducible. We co-transformed it with our plasmid and induced subcultures from an overnight growth with varying IPTG levels. We used three biological and three technical repeats and used a BMG CLARIOSTAR platereader to obtain GFP readings over the course of 10 hours in order to obtain end-point fluorescence and dynamics of the reaction.
After removing anomalous results and calculating the average and standard error, we were able to plot a graph comparing the non-quenched strain, quenched strain, and quenched-strain with the TEV-protease plasmid co-transformed. The end-point analysis showed that the induction of the TEV protease did indeed relieve quenching of the fluorescence. The levels of unquenched and Quenched+TEV protease did not differ significantly for IPTG levels of over 5µM, and even at 0µM the level of fluorescence was significantly higher in the strain with the TEV plasmid. These observations can be explained by analysing the dynamics using the time-course graph.
As can be seen on the above graph, until 200 minutes the strains appear to be acclimatising, and then induction begins to happen, this section of the graph is expanded below. From this graph it is clear that the level of induction does have an effect. The steady-state level of TEV protease produced is presumably different, and this leads to different rates of increase of GFP. Induction with only 5µM IPTG leads to a higher difference in fluorescence at 400 than at 600 minutes. The uninduced strain does not increase above the quenched strain until after 400 minutes, when it begins to climb. One interesting thing to consider is if the level of the sfGFP-quenching peptide reaches steady state in the cell, as can possibly be seen from the fluorescence of the non-quenched strain. This would mean that there is a maximum possible fluorescence level, and that over time the endpoints of all three induction levels will be equal.

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

It was unfortunate that we could not characterise the TorA leader, OmpA, or SpyTag-SpyCatcher system, however due to the design of the system we needed both parts to be induced simultaneously to get this characterisation. Our difficulties in cloning the OmpA-SpyCatcher part into an expression vector prevented this.

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:

  1. The addition of a his-tag onto the quenching peptide
  2. The addition of upstream leader sequences (TorA and SpyTag)
  3. The substitution of GFP for sfGFP

We have therefore taken another step towards the development of an assay for protease cleavage in OMVs. This could either be an assay to check for the localisation of a protease to the OMV or to see if the vesicles have been lysed by adding the protease to the media.