This construct was used by the TU Eindhoven 2023 team to form a hydrogel extracellular as well as intracellular in E.coli BL21 cells. The goal of this was to stop bacteria from dividing, however many more applications can be thought of for this part. Inspiration for this part was taken from Gradišar et al.^[1] where they used Leucine zippers to form heterodimers. We wanted to use these zipper domains in a similar way that Fernández‐Colino et al.^[2] used Leucine zippers together with ELPs to form a reversible, injectable hydrogel. Furthermore, we noticed that Contreras-Llano et al.^[3] were able to form a hydrogel inside of cells, however not with a protein based approach. We wanted to combine these ideas and form a protein based-hydrogel inside of E.coli and use these bacteria for therapeutic applications. However, with the hydrogel itself, many more applications can be thought of that we wanted to explore as much as possible so that other teams can make use of it in the future too. Below you can read more about the experiments we did to characterize this part and about the molecular dynamics simulations that we did.

A schematic overview of what the part looks like is shown in figure 2. It is simple to ligate other pieces of sequences together to make different constructs, with either different lengths of ELPs, different amino acids or other gBlocks at their end.

General applications

ELPs are protein polymers derived from human tropoelastin. One of their key features is that they exhibit a phase separation that is often reversible whereby samples remain soluble below their transition temperature (T_t) but form coacervates above T_t^[4]. They have many possible applications in purification, sensing, activation, and nano assembly. Furthermore, they are non-immunogenic, substrates for proteolytic biodegradation, and can be decorated with pharmacologically active peptides, proteins, and small molecules. Recombinant synthesis additionally allows precise control over ELP architecture and molecular weight, resulting in protein polymers with uniform physicochemical properties suited to the design of multifunctional biologics. As such, ELPs have been employed for various uses including as anti-cancer agents, ocular drug delivery vehicles, and protein trafficking modulators^[4].

Currently ELPs are also being explored in the formation of hydrogels and tissue engineering applications. We are the first iGEM team to use ELPs for the purpose of formation of a hydrogel and have made many different parts while investigating the possibilities. This part turned out to be the most sucessfull. The reversible behaviour of ELPs is even suitable for injectable hydrogels for example^[2]. We think this is a part that can have many applications inside as well as outside of cells and we hope that many other iGEM teams will make use of it in the future.

Construct design

The construct consists of ELPs and two different Leucine zippers that have affinity for each other. In general, ELPs have hydrophilic and hydrophobic domains that exhibit reversible phase separation behavior that is temperature-dependent. They are made from a repeating VPGXG sequence, with X replaced by specific amino acids. This results in specific properties of the ELPs, especially related to the T_t at which the ELPs will interact with each other on the hydrophobic sites^[2]. When the temperature is below T_t, the water molecules surrounding the hydrophobic parts will go into the bulk water phase which increases the solvent entropy. This makes it possible to form interactions with other ELP molecules^[3].

As shown in figure 3, this construct has a hydrophilic region in the middle (A_[120]) and a hydrophobic region on each side of it (I_[60]). At the ends the Leucine zippers Z1 and Z2 are located for stronger interactions between the ELPs. Leucine zippers consist of a repeating unit that forms an alpha helix. Two Leucine zippers together form ion pairs between the helices, which causes association^[4]. These stronger and reversible interactions make them useful in the formation of a hydrogel at a specific T_t. In the end, the hydrogel is formed with electrostatic and hydrophobic interactions between the ELPs.

The goal of our project specifically was to use this part to stop the cell from dividing and to co-express a therapeutic protein (Interleukin 10).

Another part that is often mentioned on this page is BBa_K4905008. This part was often used as a control. The ELP domains of the protein is the same as for the part on this page, however, part BBa_K4905008 has two Leucine zippers that are not complementary, which means that the interactions between the proteins will be weaker and this way, it could be studied what the effect of only the ELP triblocks was. This control part will be reffered to as Z2-A120-Z2 on the rest of this page. Lastly, another protein that can be seen a lot on this page is abbreviated as A120, which is the same sequence as the part on this page, but without the Leucine zippers. So it is part BBa_K4905001 ligated to itself. This protein also serves as a control group in many experiments.

Results

Protein expression and purification

This part was expressed in BL21 E.coli. The full plasmid that the DNA was cloned into can be seen below in figure 4. To check its expression we ran an SDS-PAGE gel of which the results can be seen in figure 5. The bands on the gel showed up where we expected, so this means that protein expression was succesfull.

Characterization

Hydrogel formation

First, we wanted to find out if we could use our part to form a hydrogel. All gels were formed by resuspending the ELPs in MilliQ (MQ) at different weight/volume%(w/v%). The Leucine zipper containing ELPs were expected to instantaneously form a gel upon increasing the temperature above T_t.

We saw that for both Z1-A120-Z2 and Z2-A120-Z2, a viscous fluid formed upon dissolving the ELPs in MQ water at 4°C. Subsequently, after warming the solution to room temperature, the liquid became very turbid and a gel started to form. Images of the gels can be seen in figure 6.

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 ( BBa_K4905008) 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.

Transition temperature determination with UV-Vis

The transition temperatures (T_ts) Z1-A_[120]-Z2 and control Z2-A_[120]-Z2 were determined with the use of UV-visible spectroscopy. Solutions of 5 and 20 μM were made in 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 7).

When the control protein Z2-A_[120]-Z2 crosses the T_t at 21°C, aggregates form 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 become stronger, which further increases 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. We think the interaction between Z1 and Z2 stabilizes the aggregates, forming into a branched network of ELPs, even at low concentrations of 5 μM.

Absorbances were also measured upon cooling the solution back down. Once the solution goes below T_t, the absorbances of returned back to base levels, indicating that the ELPs can be reversibly precipitated and resuspended again (Figure 8) at these low concentrations.

Dye release from hydrogel

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 9 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 (0.05 mg/mL), 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 10, 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 with limited data points. 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.

CCK8

We wanted to find a way that we could measure the amount 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^[5].

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 ^[6]. 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. A schematic representation of the experiment can be seen below in figure 11.

For this assay, it was nessecary to measure the OD₄₅₀ with a plate reader. This can directly be related to the OD₆₀₀^[5]. For this relationship, the calibration curve in figure 12 was made.

Ampicillin resistance

Three different conditions were tested over a period of time. Namely; no (protein) expression, ELPs with no cross linkers (A120), and ELPs with Leucine zippers (Z1-A120-Z2). Samples were taken at 12h, 20h, and 36h of protein expression. Before incubation ampicillin, samples were diluted to at a baseline of OD₆₀₀ = 0.5. They were then either incubated with ampicillin (1 mg/mL) or without for 1h at 37°C. The results are summarized in Table 1 and Figure 13.

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. 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 due to the fact that 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 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.

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. 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 14, 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 dilution 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 15) and the number of colonies was counted using the colony counter tool from ImageJ. The results of which are summarized in Figure 15. As expected, the wild-type E. coli treated with ampicillin did not form any colonies, whereas untreated bacteria formed several hundreds of colonies. Samples containing A120 and Z1-A120-Z2 both showed a significantly smaller amount of colonies.

Microscopy (FRAP)

Characterization of the ability of our part to form a gell inside the cell with the use of microscopy required us to design a model protein which could interact with the hydrogel, and which we could visualize under the microscope. We decided to use VPGIG_[60]-mNeonGreen (part BBa_K4905016), since the hydrophobic ELP part of the protein is able to make hydrophobic interactions with the gel. Because of these interactions, we expected that the proteins would all localize in the same places in the cell. We cloned the gene fragment into a pBad vector under the control of an arabinose promotor, to allow for orthogonality between this vector and the pET24a(+) vector encoding the zipper-ELP construct. Success of cloning was verified by a double digestion of the plasmids as well as sequencing.

However, to our surprise, the sequencing results indicated that instead of a VGPIG_[60] fragment, the mNeonGreen was attached to a VPGAG_[3]VPGGG_[2][12] fragment, which is hydrophilic, relative to the VPGIG_[60] fragment. This is likely due to a labeling mistake by a previous user of the plasmid. Due to a lack of time, we decided to continue using this construct in our microscopy experiments as a model protein to monitor the diffusion of proteins within the gelated bacteria.

Figure 15 shows two samples before and after photobleaching. Following bleach of E.coli which co-expressed Z1-A120-Z2 and and [A3G2]₁₂-mNeonGreen, a gradual recovery of fluorescence could be observed up to 37.5 seconds post-bleach (Fig. 16A, Fig. 17B). The control, containing only [A3G2]₁₂-mNeonGreen, showed a virtually instantaneous recovery, as evidenced by Fig 16B, Fig 17C.

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 t_1/2 = ln(2)*τ for each data set which is summarized in Figure 15. 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.

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

To further characterize the gelated versus non-gelated bacteria, we used a DNA staining, Hoechst (Figure 18). 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.