Revision as of 11:57, 27 October 2017

Demo

Here we show how we brought individual lab projects together and how we implement them in our device! We performed experiments in which we show that our cells are still viable after drying (which means that they can safely be shipped and still work properly), and that we can measure fluorescence in blood serum! Furthermore we combine the Signal Transduction and Specific Visualization modules to directly measure antigens by coupling the affinity molecule, Cpx signal transduction and Bimolecular Fluorescence Complementation (BiFC) specific visualization.

Cell Drying

In order to ship our bacterial system to the local health care centre (and to be used in the field later) we need to dry the cells in order to be able to ship them safely and keep them viable. We air-dry cells in a clay-matrix (Kaolin) [1] in which the cells can safely be shipped. Here we show that our cells stay viable after drying and adding clay!

Figure 1: placeholder

Cell Viability and Fluorescence in Blood Serum

Next up, we need to know if our cells can survive and function properly when we add a blood sample to measure antigens. We took several steps to investigate this. First, we analyzed if Escherichia coli could grow in (dilutions of) horse blood serum. To do this, we grew cultures with several ratios of Lysogeny Broth (LB) to horse blood serum overnight. The results can be found in Figure 2.

Figure 2: placeholder E. coli is grown in LB with added horse blood serum in different ratios. Cells were grown overnight in 2 mL cultures and the optical density at 600 nm was measured after 18 hours.

We show that E. coli is able to grow in horse blood serum concentrations up to 75%! This means that our cells would be viable when a small amount of growth medium is added before measuring, which prevents a big dilution of the antigen and, subsequently, a lower fluorescent signal.

In addition, we tested if fluorescence can be measured in blood. We grew E. coli K12 containing eYFP (BBa_K2387003) in the pSB1C3 plasmid BBa_K2387003 overnight in LB. These cells were centrifuged and resuspended in 1 mL of LB with added horse blood serum (the same dilutions were used as in the viability test above) and YFP was matured at 30 °C. Fluorescence was measured after six hours.

Figure 3: eYFP fluorescence measured in E. coli resuspended in several LB to horse blood serum ratios. eYFP was excited at 512 nm and measured at 528 nm. Measurements taken after 6 hours.

Here we show that fluorescence can be measured in all blood serum dilutions, whereas the negative controls containing no eYFP show negligible fluorescence!

Improving the Fluorescent Signal

We visualize antigen binding using the Cpx pathway by fusing split fluorophores to interacting proteins. Through a combination of wet- and dry-lab work, we found that a system based on CpxR dimerization (link MI page)yields the best results using bimolecular fluorescence complementation (BiFC) (Figure 4). We used eYFP, split after amino acid 154, as the reporter. This is a commonly used fluorescent reporter in BiFC [2].

We aim to improve this reporter, both in signal intensity and response time. During our “Fluorescent Protein” project we tested a number of fluorescent proteins, of which mVenus showed the shortest maturation time. Furthermore mVenus is designed to have a fast and efficient maturation time [3], exactly what we need!

Also, our Cpx pathway model integration (LINK) showed that several interactions of the Cpx pathway visualization can be improved, of which using a fluorescent protein with a decreased maturation time was the most feasible to attempt in a laboratory setting.

We fused mVenus-termini to the C-terminus of CpxR(link) in the same fashion as we did with eYFP Link results Bart and transformed this to E. coli K12. Experiments with mVenus were performed using the same protocol with optimal induction and activation parameters used during experiments with eYFP, and can be found here (LINK).

Figure 5: placeholder CpxR dimerization visualized using mVenus, with a L-arabinose concentration of 0.2% w/v and different activator (KCl) concentrations over time.

The results show that usage of mVenus over eYFP as a reporter protein increases the produced fluorescent signal some ten times! Unfortunately, the background signal also increases a lot, which means we lose specificity of our response. We hypothesize that the maturation rate of mVenus is too high, which means that many non-specific interactions become irreversible, leading to high fluorescent signals even when no activator is present. This means that mVenus is not a suitable candidate to visualize antigen binding within our diagnostic.

During this project, more reporter proteins were tested. Unfortunately we didn’t have time to test these in the CpxR dimerization setup. At this moment, we recommend testing sfGFP (link) as a reporter for antigen binding. We found that sfGFP is thermostable, i.e. it matures efficiently at high temperatures, while still being one of the fastest and brightest reporters we tested. You can check these experiments here(LINK JOSE).

Phase 2: Testing model propositions in the lab

To test these hypotheses, we created constructs in which we coupled split eYFP halves to CpxA and CpxR respectively and placed them under control of the inducible araC/pBAD promoter. We transformed E. coli K12 with these constructs. The Cpx system was activated with the known activator KCl in different concentrations to mimic different antigen levels at t = 20 min. Here (link) an extensive overview of the performed experiments can be found!

We quickly found out that the systems based on CpxA-CpxR interaction did not generate a clear fluorescent signal, which matches the prediction of the model! Furthermore, we show that visualizing CpxR dimerization with BiFC is indeed a viable option. Because we put the CpxR-eYFP-termini construct under control of the inducible araC/pBAD promoter we were able to test hypothesis 1: The strongest signal will be obtained when CpxR expression is maximized. We found out that this is indeed true. You can check this result here (Link Bart results).

We then set to test hypothesis 2: The strongest signal will be obtained when Cpx activation is maximized. In figure 3 we show that this is also true! We mimic antigen binding by adding a known activator of the Cpx pathway, and by increasing its concentration the fluorescence intensity also rises!

We take this data back to the lab to further improve our computer model!

Directly Visualizing Antigen Binding

Here we test a direct coupling of the projects “Signal Transduction” and “Specific Visualization”, where we express the whole detection system in one cell, from affibody to BiFC.