Part:BBa_K3425101:Experience

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Applications of BBa_K3425101

Dimerization of DBD-CaFF upon caffeine binding triggers mRFP1 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. You can read more about this assay <a href="https://2020.igem.org/wiki/images/9/93/T--UofUppsala--AutumnLab.pdf"> here</a> (Pages 1 and 2 of the notebook). 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 the stationary phase, the cells show induction of mRFP1 reporter expression from concentrations as low as 1 μM, reaching saturation at 20 μM (Figure 1D).

Cells were cultured and induced with caffeine on the same conditions as mentioned before, but on a higher volume of 5 mL. The cells were pelleted and the supernatant discarded. The pellet in all samples showed similar weight, showing that the amount or size of cells does not account for the increase in fluorescence (data not shown). The cells containing only the plasmidpSB1C3+pCadBA-mRFP1 do not seem to express the fluorescent protein at all, and the gradient of mRFP1 expression at different concentrations can be seen in Figure 2. This proves that DBD-CaFF is necessary for expression of the reporter gene, and that the presence of caffeine regulates the strength of the expression.

<img class="mx-auto" src="" width="100%" alt=""> <figcaption>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.</figcaption> </figure>

 <figcaption>Figure 2. Pelleted cells of DH5α containing plasmids pSB3K3+DBD-CaFF and pSB1C3+pCadBA-mRFP1 on R1 and R2, and only pSB1C3+pCadBA-mRFP1 on the negative control. The strains were used in the caffeine assay at different concentrations of caffeine (on top of each tube) and the pictures were taken  in (A) natural light and (B) UV 366 nm.</figcaption>

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<p>The experimental results are encouraging, but it is problematic that every time we change one of the components of the system we need to titrate the sensitivity of the biosensor. Some of the variables and equilibrium constants of the system are known experimentally or can be estimated after fitting the model with the experimental data. A model that allows us to predict the level of reporter expression reliably would be invaluable to functionalize the NANOFLEX system in biosensor applications and to accelerate the development of improvement on the system. We have used a model using stochastic differential equations (SDEs) for the simulation of mRFP1 production shown in the previous section of the proof-of-concept. The stochastic model formula can be seen below.

Stochastic model formula

R_dimer_pro_syn:

dimercaff + free > dimerpromoter

k1*dimercaff*free
R_dimer_pro_deg:

dimerpromoter > dimercaff + free

k2*dimerpromoter
R_mRNA_syn:

dimerpromoter > mRNA + dimerpromoter

kmRNA_syn*dimerpromoter
R_mRNA_deg:

mRNA > $pool

kmRNAdeg*mRNA
R_Prot_syn:

mRNA > Protein + mRNA

kProtsyn*mRNA
R_Prot_deg:

Protein > $pool

kProteindeg*Protein
R_MatureProtein_syn:

Protein > MatureProtein

kProtmat*Protein
R_MatureProtein_deg:

MatureProtein > $pool

kProtmat_deg*MatureProtein

The formulas above describe the stoichiometries and rates of each reaction in the model.

We can regulate the estimated amount of caffeine in the model by changing the dimercaff variable, since the Kd of dimerization is much higher than the Kd of caffeine binding <a href="#(2)">(2)</a>. The leakiness of mRFP1 expression is accounted for by maintaining background minimum level of dimercaff.

A limited number of promoters was set, which are categorized into free promoters (variable free) and DBD-CaFF-bound promoters (variable dimerpromoter). In the case of the backbone pSB1C3 the literature shows that it has a copy number per cell of between 100 and 500. We assume in our model the amount per cell as 100 promoters in total.

Values of 4, 472, and 4720 copies per cell of caffeine were chosen because they correspond to the concentrations tested in the lab, 0.1µM, 1µM and 10µM.

The simulations were run at values of K1 of 0.001, 0.01 and 0.1 because this variable is not known experimentally (Figure 3). Only a few of them will be presented here as relevant to the proof of concept. At concentration 4 units of caffeine per cell, the K1 value determined the maximum level of mature protein until a maximum at K1 = 0.1 with about 600 copies per cell. There is a reduction in that result at K1 = 1 with 500 copies per cell (data not shown). At 472 and 4720 the system was not responsive to changes in the value of K1, indicating saturation of the system. The results at K1 = 0.1 mirrors the behaviour observed in the empirical data within an order of magnitude for the concentration that causes saturation of induction.

<img src="" width="100%" height="" alt=""> <figcaption>Figure 3. (A) Stochastic model at dimercaff = 4, K1 = 0.001 (B) dimercaff = 4, K1 = 0.1 (C) dimercaff = 472, K1 = 0.1 (D) dimercaff = 4720, K1 = 0.1</figcaption>

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NANOFLEX can reliably respond to a complex sample containing caffeine in a predictable manner

The caffeine assay was also performed using actual coffee in order to prove the detection of caffeine within a complex sample (Pages 3 and 4 of the notebook). 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 <a href="#(1)">(1)</a>. Thus, one cup of coffee has a concentration of 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 the 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 4A-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 an extremely high standard error and technical variation, and therefore it was taken into a separate graph so that its error bars do not block all the others (Figure 4E).

<img src="" width="100%" height="" alt=""> <figcaption>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).</figcaption>

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Induction of expression of the reporter was observed according to the expected results (Figure 5A-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 than 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 <a href="#(2)">(2)</a>. In any case, these results only confirm that the system is responsive to caffeine and sensitive to the concentration of caffeine in the sample.

<img src="" width="100%" height="" alt=""> <figcaption>Figure 5. (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 the stationary phase. The concentrations of caffeine are approximated from the information available to the consumer.</figcaption> </figure>

Lyophilization of NANOFLEX is possible for potential storage and delivery

<p>E. coli cells containing pSB3K3-DBD-CaFF and pSB1C3-pCadBA-mRFP1 were lyophilized according to the protocol (Figure 6). 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.</p>

 <p><img src="" width="100%" height="" alt="">
 <figcaption>Figure 6. (A) Lyophilized cells on an eppendorf tube (B) Previously lyophilized cells grown after 1 week at 4ºC after addition of caffeine. </figcaption>

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