Formation and characterization of intracellular hydrogel

UV Vis

The transition temperatures (T_ts) of our constructs Z1-A_[120]-Z2 and Z2-A_[120]-Z2 were determined by UV-Vis spectroscopy. Solutions of 5 and 20 μM were dissolved in cold MQ water. Absorbances were measured at 350 nm. T_ts were determined to be 23.5°C and 21°C for both constructs, respectively (Figure 1). This is remarkable, since the expectation was that the construct containing Z1 and Z2, two Leucine zippers that are be able to dimerize, would have a lower T_t, with the reasoning that the interaction of these domains would cause the ELPs to become insoluble at a lower temperature. We suspect these unexpected results might be due to the fact that because of the zippers, there might be less possibilities for the ELPs to interact with eachother. This could cause the T_t to go up instead of down like we expected.

When Z2-A_[120]-Z2 crossed the T_t at 21°C, aggregates formed, leading to a sharp increase in absorbance. The absorbance then drops, which might be explained by the non-complementary two zipper domains repelling each other, leading to disassembly of the aggregates. Upon further increasing the temperature, the hydrophobic interactions became stronger, which further increased the absorbance, until finally even the hydrophilic block becomes insoluble above 50°C.

Z1-A_[120]-Z2 shows a different temperature dependent behavior compared to Z2-A_[120]-Z2, in that it shows a smaller increase in absorbance when it crosses T_t of 23.5°C, indicating the presence of smaller nanoparticles. It seemed that the nanostructures were stabilized as no further changes in absorbance could be seen up to 50 ºC. From this result, we could conclude that Z1 and Z2 might interact and stabilize the structures while Z2-A_[120]-Z2 repelled each other which has a destabilizing effect. The stabilization might be beneficial for creating stable hydrogels above 23.5 ºC.

Absorbances were also measured upon cooling the solution back down. Once the solution goes below T_t, the absorbances returned to base levels, indicating that the ELPs can be reversibly precipitated and resuspended again (Figure 2) at these low concentrations.

Hydrogel formation

After determining the T_t, the next step was to test if the ELPs could form a hydrogel. All gels were formed by resuspending the ELPs in MilliQ (MQ) water at different w/v%. The Leucine zipper-containing ELPs were expected to instantaneously form a gel upon increasing the temperature above T_t, as has also been reported in the literature^[2]. The ELPs containing FRB and FKBP12 were expected to form a gel after the addition of rapamycin.

The hypothesis was that the zipper-containing ELPs would also show reversible behavior upon formation of the gel, as is also seen in the research of Colino et al. (2015)^[2]. However, having a non-reversible gel has its obvious advantages too, as the samples do not need to be kept above T_t in order to preserve the gel. Therefore, bacteria which become gelated might not have the ability to divide once the gel has formed, whether this be at low or high temperatures. This would also be a benefit when it comes to biocontainment, since cells would not again start dividing when the temperature is lowered. This point has also come up in our conversations with RIVM..

We saw that for the constructs containing the Leucine zippers, a viscous fluid formed upon dissolving the ELPs in MQ water at 4°C. Subsequently, after warming the solution to room temperature, it became highly turbid and a gel started to form. Images of the gels can be seen in Figure 3.

The construct containing Z1-A120-Z2 formed a gel at 5% and 10% (w/v%) as described above. Upon cooling the gel down to 4°C again, the gel remained intact. As a control, Z2-A120-Z2 was also dissolved to form solutions of 5% and 10% (w/v%). This construct too, was able to form a gel instantly upon heating to RT. However, in contrast to the Leucine zipper-ELPs, the gel disassembled again when the solution was cooled to 4°C. This indicates that the latter of the two constructs shows reversibility, which is expected when the ELPs are brought below their T_t, while the former construct does not show this reversibility. This indicates that at high concentrations, the interactions are not completely reversible.

As for the constructs containing FRB and FKBP12, we attempted creating gels in the same conditions as for the Leucine-zipper. FRB-A120-FKBP12 was dissolved in MQ water to a final concentration of 10 w/v%. To induce crosslinking, rapamycin was added both at 4°C and 20°C at a 1:1 molar ratio with the ELPs. However, to our surprise, the solution did not form into a hydrogel, even when the solution temperature was brought to RT.

Since we managed to form gels with the Leucine zipper ELPs, we made the decision to abandon the constructs involving rapamycin. In addition to the hydrogel not forming, these constructs would have been harder to use in vivo, since rapamycin is an anti-cancer drug^[3]. This might have led to unwanted side effects upon lysis of the bacteria when they are located in the gastrointestinal (GI) tract.

CCK8

We wanted to find a way that we could measure the number of cells that we can stop from dividing with our hydrogel and whether they would stay alive. We came across Cell Counting Kit 8 (CCK8). This is an assay that is commonly used to determine the number of alive cells in a sample. The kit uses a highly water-soluble tetrazolium salt, WST-8, which produces a formazan dye upon reduction in the presence of an electron mediator. The amount of formazan generated by dehydrogenases is directly in proportion to the number of living cells. Yang et al. (2021) have demonstrated that this assay is also suitable for the detection of living E. coli^[4].

We used this assay to test whether our hydrogelated cells would be more resistant to several conditions, including incubation with ampicillin and freeze-drying. Our biggest goal was to find out if cells would stop dividing, but stay functional with our hydrogel. To test this, we came up with the idea of combining the CCK8 assay described above with the antibiotic ampicillin. Ampicillin prevents the synthesis of the bacterium’s cell wall. It does this by binding to enzymes necessary for the formation of the cell wall^[5]. This means it only kills bacteria when they divide. Our hypothesis is that if our hydrogel prevents the bacteria from dividing, they should be affected less by the presence of this antibiotic, and therefore show a higher survival rate than bacteria that do not contain the hydrogel since these will still divide and be killed by the antibiotic. Additionally, since the concept of our treatment is to put them in a capsule which can be orally administered, the bacteria need to be able to survive freeze-drying, so that they can produce the therapeutic locally when they are reconstituted in the GI tract.

More about the protocol we followed can be found on our Experiments Page. A schematic representation of the experiment can be seen in Figure 5.

For this assay, it was nessecary to measure the OD₄₅₀ with a plate reader. This can directly be related to the OD₆₀₀^[4]. For this relationship, the calibration curve in Figure 6 was made.

Ampicillin resistance

Three different conditions were tested over a period of time. Namely: no (protein) expression, ELPs with no crosslinkers (A120), and ELPs with Leucine zippers (Z1-A120-Z2). Samples were taken at 12h, 20h, and 36h post inoculation in auto induction medium. Before incubation with ampicillin, samples were diluted to the same concentrations determined by OD₆₀₀ = 0.5. They were then either incubated with ampicillin (final concentration of 1 mg/mL) or without for 1h at 37°C. The results are summarized in Table 1 and Figure 7.

Ampicillin-treated E. coli that were not induced to express proteins, and which were treated with ampicillin, showed at most a WST-8 conversion of 0.71% ± 0.05% compared to cells which were not treated, as measured by absorbance at 450 nm, meaning that most treated cells were killed by ampicillin. In contrast, E. coli where expression of A120 was induced, showed a maximum conversion of 6.80% ± 0.17% after 36h of protein expression. This is likely because this construct is also able to form a gel, although less strong compared to the Z1-A120-Z2 construct, leading to an attenuation in cell division. Finally, Z1-A120-Z2 was expressed for the three-time points. An increase was observed in the conversion of substrate, most notably after 20h of expression (20.86% ± 4.14%). We had expected to see a higher percentage after 36h of expression, since at this time point, more bacteria should be gelated and thus unable to divide. The standard deviation of the samples is quite high, with this small number of measurements and large deviation, it is hard to tell whether there is really a difference between these two time points. Overall, a larger sample size could help to reduce the variability of this assay in future tests. Other explanations could be that some of the cells were over-gelated, which might have led to a premature death unrelated to the incubation with ampicillin. Additionally, cell death could also have occurred due to nutrients in the culturing medium running out. From this result, we could conclude that Z1-A120-Z2 expressing cells are the most resistant to ampicillin.

For comparison, OD₆₀₀ of all samples was also measured after incubation with and without ampicillin. When the samples were incubated with ampicillin, they all decreased in OD₆₀₀. The samples incubated without ampicillin in which ELPs were expressed show a relatively stable OD₆₀₀ compared to the sample in which no expression was induced indicative of attenuated growth. This was the only sample in which a substantial increase in OD₆₀₀ could be observed.

The OD₆₀₀ was also measured without addition of ampicillin over time for each of the three samples (No expression, A120 and Z1-A120-Z2). After 20h of expression, these were diluted to OD = 0.1 and then measured every 30 minutes. From Figure 8, it becomes clear that when no expression of ELPs was induced, the bacteria divided more rapidly than when expression was induced. These findings are in line with the results of the ampicillin treatment. It seems that the presence of the ELPs does not altogether inhibit division, but it does slow it down, which seems to be the reason why they are killed slower upon the addition of ampicillin. However, it is hard to draw a conclusion as to whether the attenuation of bacterial division is driven by the formation of a gel inside the cytosol, or simply because of the crowded environment inside the cell, caused by the presence of a high amount of protein. Therefore, additional controls need to be added in future experiments, like samples in which a mono- or diblock ELP is expressed, in order to account for molecular crowding. Furthermore, a sample where a completely different protein is also interesting, to see what the effect of protein expression is on cell division.

These results were compared to the classic colony counting method. From each sample, a diltion of 10⁵ was made and then plated out on an LB-agar plate. The plates were incubated at 37°C overnight. Images of the plates were taken (Figure 9) and the number of colonies was counted using the colony counter tool from ImageJ. The results of which are summarized in Figure 9. As expected, the wild-type E. coli treated with ampicillin did not form any colonies, whereas untreated bacteria formed several hundreds of colonies. In case of A120 and Z1-A120-Z2 expressing bacteria on agar plates without amplicillin, number of colonies were significantly lower indicating that bacteria growing was inhibited, consistent to our OD measurement (Figure 8). When treated with ampicillin, only the sample with bacteria expressing Z1-A120-Z2 forming colonies indicating they are more resistant. In summary, we can conclude that these bacteria are ampicillin resistant while growth was attenuated, likely because hydrogel formation due to ELP production. 142 colonies survived in ampicillin compared to 297 colonies that formed in ampicillin free medium.

Freeze-drying

From conversations with several experts, we got the advice to freeze-dry the bacteria as is described in more detail on the Human Practices Page.. Freeze dried bacteria are easier to store at milder conditions, which will help us distribute cELPro to even the hardest to reach places in the world. Since our ML-I Biolab did not contain a freeze-drying setup, we assembled one ourselves, as is decribed in Week 38 of the Notebook. We did this in one of the fumehoods in the biolab and we attempted to freeze-dry six different types of sample (Figure 10A). Three for each of the different conditions that were also tested previously (no expression, A120 and Z1-A120-Z2) and these same three, but with 100 mM trehalose added. This is a sugar which protects the bacterial membrane during freeze-drying^[7], and was therefore meant to be used in the samples for a positive control. However, even after running the setup for over 8h, some samples unfortunately did not sublimate, and since this setup required us to refill the cold trap with liquid nitrogen every couple of hours, we could not leave it overnight. Therefore, only two of the samples, Wt and Z1-A120-Z2, were dried relatively well. These were then reconstituted and subsequently assayed using CCK8, of which the results can be seen in Figure 10. Using the calibration curve made for this assay (Figure 6), the OD₆₀₀ values were calculated from the measured OD₄₅₀ values. These were then divided by 0.5, the original OD₆₀₀ at which the samples were made, to obtain the final survival rate of the bacteria.

A

Sample	Success?
No expression	Yes
No expression + 100 mM trehalose	No
A120	No
A120 + 100 mM trehalose	No
Z1-A120-Z2	Yes
Z1-A120-Z2 + 100 mM trehalose	No

B

Small molecule diffusivity

To determine the relative ability for the diffusion of small molecules through our hydrogels, we made gels at two different weight-to-volume percentages containing the fluorescent dye Rhodamine B. Finding out whether small molecules can gradually diffuse out of the hydrogel might be interesting for future applications, a drug could by incorporated for instance.

First, a calibration curve was made to determine at which concentration Rhodamine B should be added to the gel. Figure 11 shows a linear relationship up to 26 μM of rhodamine B. We decided to make the gels with a Rhodamine B concentration of 104 μM, since the amount of dye diffusing out of the gels was expected to be far lower than this amount. A 5% gel as well as a 10% gel were tested. Our hypothesis was that diffusion through the 5% gel would be faster than when a higher weight percentage of the ELPs was used.

The gels were made at a volume of 100 μL. They were then brought to RT and washed with 1.5 mL of warm MQ. Then, 1 mL of warm MQ was pipetted on top of the gels, and they were incubated for up to 8 hours at RT, with samples of 100 μL being taken every 2 hours. After measuring the fluorescence intensity, the values were corrected for the reduction in volume upon taking the samples. From Figure 12, it becomes clear that the dye diffuses faster from the 5% gel than from the 10% gel. However, more experiments are needed to truly find the rate of diffusion, it is hard to draw a conclusion now with only few data points. It is also important to note that the last three timepoints gave measured values that were higher than the linear part of our callibration curve. Repeating the experiment with a lower concentration of dye would therefore be disirable.

The faster diffusion rate of the 5% gel is in line with our hypothesis that a lower weight percentage gel is able to release small molecules faster than a 10% gel. Since Rhodamine B is a much smaller molecule than a cytokine like IL-10, it would be interesting to see how the diffusion of such a larger molecule would be affected by our gels. The weight percentage of the gel might therefore not only affect the division rate of the bacteria, but it can also play a role in the release rate of the therapeutic.

A

B

Microscopy

The bacteria containing Z1-A120-Z2 and [A3G2]₁₂-mNeonGreen were used to determine if diffusion throughout the cell was affected by the formation of the gel. This was done by using fluorescence recovery after photobleaching (FRAP). We hypothesized that once the gel is formed intracellularly, the diffusion rate is negatively affected due to an increase of viscosity of the cytosol.

Figure 13 shows two samples before and after photobleaching. Following bleach of E. coli co-expressing Z1-A120-Z2 and [A3G2]₁₂-mNeonGreen, a gradual recovery of fluorescence could be observed up to 37.5 seconds post-bleach (Figure 13A, Figure 14B). The control, containing only [A3G2]₁₂-mNeonGreen, showed a virtually instantaneous recovery, as evidenced by Figure 13B, Figure 14C, because the fluorescent molecule might not be hindered by crowded environment formed ELPs.

The raw data was corrected for photobleaching during post-bleach image acquisition and normalized to the intensity pre-bleach. The data was then fitted with equation 1, Where I(t) stands for the normalized fluorescence intensity at time t, A is the mobile fraction, t is time and τ is the time constant^[7]. This was done for each of the measurements, resulting in four graphs for the co-expressed sample and two graphs for the control. Using the equations, the recovery half-life (τ_1/2) was calculated, using (τ_1/2) = ln(2)*τ for each data set which is summarized in Figure 14. For a more detailed description of the FRAP experiment, see the Protocol Book.. This experiment shows that proteins are distributed throughout the whole cell and that the diffusion rate becomes lower when our ELP constructs are expressed. It is impossible to exactly determine the intracellular concentration of ELPs, and thus to make a comparison with the gels made extracellularly. However, it would be worthwhile doing these FRAP measurements on more samples in the future where, just as was done for the CCK8 assays, protein expression is induced for a series of different timepoints to see the effect on diffusion. In addition, control with non-gelated ELPs would be more insightful.

I(t) = A(1-e^(-t/τ))+c (1)

A

Measurement	τ1/2 (s)
1	23.9
2	7.0
3	16.5
4	11.7
Average + stdev	14.8 ± 6.2

B

C

To further characterize the gelated versus non-gelated bacteria, we used a DNA staining, Hoechst (Figure 15). We saw that a large number of gelated bacteria showed a clustering of DNA in the center of the cell, whereas for the control sample, the DNA was more homogenously spread throughout the cell. This might indicate that the DNA in gelated cells is not as accessible to replication machinery and that therefore division of the cells is stopped or slowed down.