<

Results
(DNA-based System)

Introduction

In order to demonstrate the feasibility of our DNA-based system for detecting cruzipain, we designed and cloned four parts into the pSB1C3 vector:

mCherry-TEV Protease-His tag
TetR(With an engineered TEV protease cleavage site)-CFP-His tag
TetR(WT)-CFP-His tag
pTet-RBS-eYFP

From our modelling of this system and our research into cruzipain levels in the blood, we estimated the level of cruzipain that would be present. We then calculated the optimum levels of TetR and our pTet construct that would limit false positives and negatives in such a system. We added fluorescent reporters (which did not have significant overlap in their spectra) to our three parts in order to visualise their levels, and hence determine the levels of inducers we needed to accurately replicate our in silico model in vivo.

Shipping Vector Cloning

A more in-depth description of this stage of our project can be found on the Shipping Vector Cloning results page. For the DNA-based part of our project, we cloned the four parts into the shipping vector.

We then used quick-change PCR to perform site-directed mutagenesis on our TetR parts and the pTet part. In the TetR parts we had mistakenly included a stop codon at the end of the CFP coding sequence before the His-tag, so although this would not have affected the workings of the part it would have meant we wouldn’t be able to purify it. The pTet-eYFP had an illegal restriction site so we used Quick-Change PCR to make it compatible with registry standards.

This, as the name suggests, was a quick and easy procedure and we recommend it to any team that is in a similar situation. The protocol can be found on our protocols page.

Expression Vector Cloning

In order to perform full characterisation of our parts we needed TEV Protease, TetR, and pTet-eYFP in the same cell. As the likelihood of a triple transformation was very low, we decided to clone both the TetR and the pTet-eYFP into the same plasmid. We then would only need to do a double transformation of this and the TEV protease plasmid. We selected pQE-60 and pBAD-33 as they had compatible origins, different antibiotic resistances, and were induced by two different and easily-obtainable inducers, IPTG and arabinose respectively.

We designed primers for two 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 version of the mCherry-TEV Protease-His tag, TetR(With an engineered TEV protease cleavage site)-CFP-His tag and TetR(WT)-CFP-His tag parts without fluorophores. This was ensure the fluorophore did not affect the activity.

We were successful in doing this for mCherry-TEV protease-His tag, as well as the no fluorophore version, and pTet-eYFP into pBAD33 and pQE-60 respectively. We further characterised the mCherry-TEV protease-His tag and pTet-eYFP.

For the other parts in our system, we unfortunately did not obtain a correctly sequenced version in the expression vector, despite obtaining the correctly sized bands in the test digest agarose gels of our minipreped DNA.

To try and resolve this problem we did a number of things:

We repeated the cloning many times with no luck.
We thought that it could be a problem with the primers, so we tried longer primers, which still yielded no positive results.
We wondered if the vector stock has been mixed up, and we had managed to clone the part into a different vector. Therefore, we created some primers that would sequence from inside the part to out - again to no avail.
We requested the sequencing company to optimise the reaction conditions and help troubleshoot, and though this was attempted it did not give us decent sequencing data.

Due to the integrated nature of our system, it was therefore difficult to characterise the whole circuit without having every part correctly sequenced. As we couldn’t get our novel TetR engineered with a TEV protease cleavage sequence (which was central to this particular system) into an expression vector, it was particularly difficult to get data on the auxiliary parts.

TetR(with an engineered TEV protease cleavage site)-CFP-His tag

Whilst waiting for sequencing optimisation, we decided to continue with the characterisation of this part for two main reasons:

It was the final piece to complete the DNA-based system
Test digests suggested that the DNA we had miniprepped was of the right size.

In vivo - Fluorescence microscopy

We co-transformed the TetR-CFP with the correctly sequenced pTet-YFP into the same cell. The hope was to see just YFP when the cells were uninduced with IPTG and then a reciprocal relationship between CFP and YFP and induction levels were increased. An image we obtained is shown below in Figure 1.

The above image is a promising microscopy image which showed a binary, yet reciprocal expression of YFP and CFP, suggesting that our TetR could possibly be functioning as designed to repress pTet eYFP. However, this wasn’t consistently reproducible.

In vitro - Protein purification and TEV protease assay

In parallel to the fluorescence microscopy, we attempted to purify the engineered TetR based on a protocol provided in a paper by Mortlock et.al.

We were helped greatly in this regard by Associate Prof. Maike Bublitz, who 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 Oxford Department of Biochemistry.

An SDS-PAGE gel showed that we had eluted an array of non-specific proteins. However there was a thick band near 50kDa which is the size of TetR+CFP, suggesting that we had been partially successful. Due to the impure nature of the gel, it meant it could be a different protein.

Figure 1: Protein Purification of Engineered TetR

If the band was our engineered TetR, it would be sensitive to TEV protease action after concentrating the protein through a 5kDa filter, which gave us a 1.56 mg/ml solution. We testedthe concentrated engineered TetR with externally added TEV protease provided by Prof. Bublitz.

We tested two concentrations of TEV protease, a 1:100 molar ratio (relative concentration of TEV protease to concentration of ‘Engineered TetR’) and a 1:10 ratio. We quenched the reaction at different time points (0mins, 60mins, 120mins, 180mins, O/N) by adding it to SDS Loading Buffer. Another SDS PAGE gel was run using these time points.

The gel below in Figure 2 shows us that there doesn’t seem to be any change in the band near the 50kDa mark.

However, interestingly at the 150kDa mark, there is a sharp band that disappears over time in 1:10 TEV protease. Seeing as there was no real result for our TetR, we decided to ignore these results and focus on the characterisation of other parts.

Figure 2: TEV protease assay

Future Work

It is not trivial to go from an idea to a fully-functioning product over the course of one summer. Therefore we have envisioned the next series of experiments that would be performed to develop our project into something that was suitable for clinical trials, incorporating the ideas of constant feedback from modelling to wet lab and vice versa to ensure an optimal system. We propose five design-build-test cycles.

Design-Build-Test 1 - Full experimental proof-of-concept

We add IPTG to induce the production of TetR, which binds to the tet operator. We would test this by looking at the level of induction needed from our construct to reduce the fluorescence. This would allow a more accurate figure for our modelling of the amount of TetR needed and could be put into our model to see if this changes any parameters going forward.

Once we have established the amount of TetR needed, we would create cells as a double transformation with a TEV plasmid, and induce expression of TEV with arabinose. TEV can then cleave the TetR.

If TetR is cleaved then YFP will start to be produced.

We can relatively quantify the amount of YFP produced, comparing different levels of induction of TEV.

Design-Build-Test 2 - Proof-of-concept system in cell lysate

Instead of inducing TetR production, we add a calculated amount of purified TetR to the system and allow it to bind. This will check that our purification doesn’t affect the efficacy of TetR.

We induce the TEV protease by the addition of arabinose, as before.

TEV protease can then cleave TetR, as before.

We can quantify the fluorescence and compare all of the data to the system in the cells, and use this to feed into our DNA system modelling. We can also optimise our lysate.

Design-Build-Test 3 - Test with cruzipain and production of TEV protease

We would then construct a plasmid with TEV protease under the tet operator and promoter, and add this to a cell lysate with an appropriate amount of TetR, as determined in the above experiment cycles.

We would then add cruzipain to our system, to check that the TetR can be cleaved by cruzipain in the same way as it was cleaved by TEV protease.

The output would be the TEV-mediated cleavage of something like a quencher-fluorophore system, such as BBa_K2450501.

If cleavage occurs, then all the above steps were successful, and we will prove that the system works for cruzipain as an input.

Further DBT cycles

A repeat of cycle 3, in a blood sample, testing our output bivalirudin

We would do the same experiments as cycle 3 but testing them in blood. We will see a positive result for our test if the blood does not clot, as this means bivalirudin has been released. We would also check that our steric hindrance works by adding the hindered peptide to a blood sample and checking it clots.

We would then repeat again, starting with a freeze-dried kit. This would check that it will be able to travel in this form, and that the addition of blood will be sufficient to rehydrate the kit. At this point we can also experimentally test to see the level of cruzipain which we can detect, and see if it matches our model.

Team:Oxford/Results DNA

Results(DNA-based System)