Team:UofUppsala/Proof Of Concept


Navigation Bar

Proof of concept


Introduction

Here we have gathered the steps leading to experimental evidence for the functionality of the NANOFLEX system. In the presence of the target antigen, the cells containing the plasmids pSB3K3+DBD-CaFF and pSB1C3+pCadBA-mRFP1 should induce the expression of the selected reporter. Only once the basic functionality is tested it’s reasonable to propose improvements for the system, like using a different nanobody or a reporter gene of faster response such as lacZ. We want to be able detect caffeine at very low concentrations to prove that the system is functional and useful in all relevant conditions. The main questions that need to be answered are:

  • Is Nanoflex reporter expression induced by the targeted antigen?
  • IDoes the device affect cell growth in any way?
  • IHow much time does it take to observe the induction?
  • ICan we model and predict Nanoflex behavior?
  • IDoes the system still work as expected while using a complex sample?
  • ICan the system be transported and stored for later use?

Results

Induction of reporter expression

The cell growth and fluorescence signal upon exposure of the Nanoflex device to the different caffeine concentrations was measured over time when using the caffeine induction assay. (Protocol) The growth curve was similar at different caffeine concentrations, showing that these caffeine concentrations were not toxic to the cells and that the stationary phase is reached at 3.5 hours of growth (Figure 1A-B).

The engineered strain of E. coli DH5α reacts to the presence of caffeine in different concentrations (Figure 1C). When 1 μM caffeine is present (blue), the levels of fluorescence are significantly higher compared to the background signal generated in the absence of caffeine (black). Higher caffeine concentrations (light blue, purple, apricot and red) cause an earlier appearance of the signal distinguishable from the background and higher fluorescence levels later on. No increase in the fluorescent levels can be seen when the results 20, 50 and 100 μM caffeine concentrations are compared, which testifies about the saturation of the system at 20 μM. During measurements, cells were cultivated in the humidity chamber of the Spark 10M microplate reader (Tecan ), at 37 ºC, using Nunclon 96 Flat Black plates (Thermo Fisher Scientific). Fluorescence was measured by measuring emittance at 611 nm after excitation at 585 nm, and normalized by the optical density measured at 700 nm after subtraction of the LB media fluorescence values. Each dot represents the average of two biological replicates and error bars are respective standard errors. After the culture reaches stationary phase, the cells show induction of mRFP1 reporter expression from concentrations as low as 1 μM, reaching saturation at 20 μM (Figure 1D).

Figure 1. (A-B) Growth curve of two biological replicates at different caffeine concentrations of E coli DH5ɑ double transformed with pSB3K3 + DBD-CaFF and pSB1C3+pCadBA-mRFP1 measured at OD700. (C) Normalized fluorescence of engineered cells at different caffeine concentrations over time. The bars represent the standard error. (D) Normalized fluorescence at 3.5 hours of growth at different caffeine concentrations.

Figure 2. (A) Formula of the stochastic model, where yt is the amount of protein at time t,yt-1 the amount of protein in the previous second and ϵ the noise. (B-H) Plot of the concentration of protein as natural logarithm through time at initial concentrations of 30, 60, 70, 80, 110 and 120 μM, respectively.

Induction experiment with a real sample

The caffeine assay was also performed using actual coffee in order to prove the detection of caffeine within a complex sample. Decaffeinated coffee was also used, since it is not completely caffeine free but it contains a lower concentration and thus should give a lower level of induction using the same amount of sample. According to Livsmedelsverket there is about 44.5 mg of caffeine per 100 mL of coffee (Reference). Thus, one cup of coffee has a concentration around 2.5 mM caffeine (C), that added using the induction assay protocol gives a final caffeine concentration of 50 μM (see notebook). Decaffeinated coffee have an approximated cup concentration of 0.1 mM (dC), and a final assay concentration of 2 μM. According to the data in figure 1, using this concentration in the induction protocol is sufficient for induction of mRFP1 expression. The coffee used for the experiment was Lindvalls Kaffe Tricole, and Gevalia Mellanrost Koffeinfritt was used as the decaffeinated control. The samples did not affect growth in any significant way (Figure 3A-D). The plots were organized as separate biological replicates to allow for easier understanding. However, the sample of replicate B that has no coffee sample at all has extremely high standard error and technical variation, and therefore it was taken into separate graph so that its error bars do not block all the others (Figure 3E).

Figure 3. Growth curves of two biological replicates, cultures A and B, both of them measured as OD700 and standard error bands from 2 technical replicates growing at several concentrations of the coffee and decaffeinated coffee extracts: (A) culture A, coffee sample (B) culture A, decaffeinated coffee extract (C) culture B, coffee sample (D) culture B, decaffeinated coffee extract (E) culture B concentration 0 μM of sample (distilled water).

Induction of expression of the reporter was observed according to the expected results (Figure 4A-B). The samples in absence of C or dC had the lowest levels of fluorescence while the other samples saw an increase in fluorescence (data not shown). The induction using a similar dilution of 1/50 C (1μM) was higher that for the dilution 1/50 dC (0.04 μM), but almost the same as dC (2 μM). The induction using the normal concentration of C was higher than that of dC. The induction of the second gave a similar value to the expected one, but it wouldn’t be unexpected if the result was different due to the fact that the nanobody that we use also bind to other molecules of the caffeine pathway like theobromine, albeit with a lower affinity (Reference). In any case, these results only confirm that the system is responsive to caffeine and sensitive to the concentration of caffeine in the sample.

Figure 4. (A) Normalized fluorescence of mRFP1 in the presence of different concentrations of coffee sample and decaffeinated coffee extract over time. (B) Normalized fluorescence of mRFP1 in the presence of different concentrations of coffee sample and decaffeinated coffee extract at 3.5 hours of culture, at the beginning of stationary phase.

TypeIIS modular replacement of nanobody

Lyophilization

E coli cells containing pSB3K3-DBD-CaFF and pSB1C3-pCadBA-mRFP1 were lyophilized according to the protocol. The cells were stored for a week at 4ºC. After that, the gel pellet was added 500 uL of LB + Km and Cm, and caffeine to a final concentration of 5 uM. The pellet was resuspended, and the cells grown overnight at 37ºC in a shaker. The next morning,mRFP1 and cell growth could be confirmed in the media, proving that the lyophilized cells stayed alive and maintain mRFP1 expression capability.

Figure 5. (A)Lyophilized cells on an eppendorf tube (B) Previously lyophilized cells grown after 1 week at 4ºC after addition of caffeine.

Conclusions

Induction of the Nanoflex system in the presence of the target antigen was observed. The results were replicable, and the induction was responsive to the concentration of caffeine. Moreover, the system maintained the expected responsiveness against a complex sample that contains many different compounds. These are encouraging results that indicate the sensitivity and specificity of the system is mainly determined by the nanobody attached to Nanoflex, with few off-target binding. The ASSURED principles are generally used for evaluating the effectiveness of diagnostic components (Reference), and here we have proven two of those requirements.

Modelling

After cell lyophilization, cell growth and mRFP1 expression was achieved after 1 week of storage. According to the literature, cells in these conditions maintain viability and functionality even after 1 year of storage. The possibility of long-term storage and transport is necessary to deliver any potential biosensor derived from Nanoflex to the end user.

Demo version on NANOFLEX

NANOFLEX is a detection device and in this section, the results showing the ability of the NANOFLEX biosensor to specifically detect caffeine in different concentrations are presented.

Detection of pure caffeine

Escherichia coli DH5alpha were engineered by introducing two plasmids as described in the [WETLAB - strain construction]. This strain was grown to the exponential phase when different concentrations of caffeine were added. The growth and the fluorescence signal were monitored over time.

  • system reacts to caffeine at 1 uM and higher
  • the level at 0 caffeine concentration is the level of noise of the system = possible causes
  • Significant difference is seen between the 1 uM, 10 uM and
  • We have shown that the differences in the fluorescence are not due to the large difference in growth rates

Figure 1. | The fluorescence signal upon exposure to the different caffeine concentrations was measured over time.

The engineered strain of E. coli DH5α reacts to the presence of caffeine in different concentrations. When 1 μM caffeine is present (blue dots), the levels of fluorescence are significantly higher compared to the background signal generated in the absence of caffeine (black dots). Higher caffeine concentrations (light blue, purple, apricot and red dots) cause an earlier appearance of the signal distinguishable from the background and higher fluorescence levels later on. No increase in the fluorescent levels can be seen when the results 20, 50 and 100 μM caffeine concentrations are compared, which testifies about the saturation of the system at 20 μM. During measurements, cells were cultivated in the humidity chamber of the Spark 10M microplate reader (Tecan ), at 37 ºC, using Nunclon 96 Flat Black plates (Thermo Fisher Scientific). Fluorescence was measured at XXX nm and normalized by the optical density measured at 700 nm after subtraction of the LB media values. Each dot represents the average of two biological replicates and error bars are respective standard errors.

Figure 1. | Detail of the differences in fluorescence levels 3.5 hours after addition of caffeine in different concentrations. The engineered strain of E. coli DH5α reacts to the presence of caffeine in different concentration

Figure 3. Growth curve of two biological replicates form figure1 and 2, growing at the same rate at different caffeine concentration = not toxic, 3.5 hours is timepoint when cells are just about to enter the stationary phase

Figure 5. (Coffee versus decaffeinated coffee) - Detail at 3.5 hours post induction again.

As Figure 11 shows, cells are about to enter stationary phase

Figure 6. (Coffee versus decaffeinated coffee) - Growth curves! two biological replicates, cultures A and B, both of the growing at several concentrations of the coffee and decaffeinated coffee extracts: culture A, coffee culture A, decaffeinated coffee extract culture B, coffee culture B, decaffeinated coffee extract culture B - "with water" - growth curve with water has extremely high standard error, technical variation, and therefore it was taken into separate graph from (C ), so that the error bars do not block all the other error bars. Error bars are technical variation, calculated from two technical replicates as standard error.

Lyophilization

Protocol

Lyophilized cell preparation:

E coli cells containing pSB3K3-DBD-CaFF and pSB1C3-pCadBA-mRFP1 were grown at 37ºC under shaking until OD600 = 0.3 (at the start of the exponential phase). The cells were pelletized by centrifuge 5 minutes at 5000 rpms, and resuspended to an OD600 = 0.5 in LB + sucrose 12%. The cells were aliquoted by adding 500 uL in 2 mL eppendorf tubes then frozen at -80ºC. After that, the cells were lyophilized for 6 hours in a SpeedVac Vacuum Concentrator. The lyophilized cells were stored for a week at 4 ºC before their use. This protocol is mostly derived from Prévéral et al, 2017.

Experiment

The gel pellet was added 500 uL of LB + Km and Cm, and caffeine to a final concentration of 5 uM. The pellet was resuspended, and the cells grown overnight at 37ºC in a shaker.

Figure 1. Lyophilized cells on an eppendorf tube

Figure 2. Previously lyophilized cells grown after 1 week at 4ºC after addition of caffeine. mRFP1 and cell growth can be confirmed.

Conclusions

Cell growth and mRFP1 expression was achieved after 1 week of storage. According to the literature, cells in these conditions maintain viability and functionality even after 1 year of storage.

References

  1. Prévéral, S., Brutesco, C., Descamps, E. C., Escoffier, C., Pignol, D., Ginet, N., & Garcia, D. (2017). A bioluminescent arsenite biosensor designed for inline water analyzer. Environmental Science and Pollution Research, 24(1), 25-32.