Difference between revisions of "Part:BBa K4905006"

Revision as of 11:51, 23 August 2023

Elastin-Like Polypeptide Triblock with Leucine Zippers

Information

This part is made up of the basic parts: Leucine zipper Z1 (BBa_K4905004), Leucine zipper Z2 (BBa_K4905005), and two times Elastin-like polypeptide sequence A[60]I[60] (BBa_K4905001). It was used by the TU Eindhoven 2023 team to form a hydrogel outside as well as inside e.coli BL21 cells.

The construct consists of Elastin-like Polypeptides (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. As shown in figure ..., this construct has a hydrophilic region in the middle and a hydrophobic region on each side of it. On both ends, the leucine zippers Z1 and Z2 are located for stronger interactions between the ELPs. This allows them to be used in the formation of a reversible hydrogel at a temperature of 37 °C.

figELP

Figure 1: Schematic representation of the composite part, an Elastin-Like Polypeptide with leucine zippers on the ends

Application

In the TU Eindhoven 2023 team project, the ELPs are, in the end, expressed inside e.coli BL21 cells. The hydrogel will be formed with electrostatic and hydrophobic interactions. At a temperature below 37 °C, the hydrophobic parts are surrounded by water molecules. This causes a low entropy state. When the temperature increases over 37 °C, these water molecules go into the bulk water phase what gains the solvent entropy. This makes it possible to form interactions with other ELP molecules [1].

When the hydrogel is formed inside e.coli BL21 cells, it prevents the cell from dividing. However, the cells remain functional. So they can still be used to express therapeutic agents, like Interleukin 10 in the project of the TU Eindhoven 2023 team.

Sequence and Features

Results

Protein expression and purification

The protein has an expected molecular weight of 105.1 kDa Organic solvent extraction, ITC

SDS-page

fig1

fig2

1. Z1-A120-Z2
2. Z2-A120-Z2
3. Z2-A100-Z2

Characterization

Massspec

Hydrogel formation

To see if a hydrogel could form, 10 wt% ELP was dissolved in cold MQ and left at room temperature to warm up and form a gel. This gel could be dissolved again when put at 4 °C overnight.

fig3

Figure 2 A ten percent hydrogel was formed inside of a mass spectrometry vial. ELP constructs were dissolved in MQ at 4 °C and left at room temperature as soon as they dissolved. Within minutes, a hydrogel started to form.

Transition temperature determination

To determine the transition temperature of the ELP constructs, different solutions of the proteins were made in PBS. Using a UV-VIS spectrometer, the absorbance of light at 350 and 600 nm was measured to find the temperature at which phase separation happens. First, the samples were heated up to find the temperature where the hydrogel forms. Later, the samples were cooled again to show their reversible behavior. Two transition temperatures can be seen, the first is where the hydrophobic parts aggregate and the hydrogel forms, the second transition temperature is where the hydrophilic blocks also collapse. Two different constructs are plotted together, Z1-A120-Z2 has complementary leucine zippers and Z2-A120-Z2 has two leucine zippers that are the same, so it acts as a control group. It can be seen that their transition temperatures differ. The transition temperature of Z2-A120-Z2 is about 17 °C and Z1-A120-Z2 has a transition temperature of around 20 °C. It can also be seen hat the Z1-A120-Z2 construct only exhibits one transition temperature, although two were expected, this might be because of the zippers already bringing the hydrophilic domains together which loses the second transition temperature.

fig4

Figure 3 Temperature dependent behaviour of the Z1-A120-Z2 and Z2-A120-Z2 ELP constructs at 20 uM and 5 uM concentrations measured at 350 nm. The temperature was varied with 0.5 °C per minute. The transition temperature of Z2-A120-Z2 is about 17 °C and Z1-A120-Z2 has a transition temperature of around 20 °C.

fig5

Figure 4 Temperature dependent behaviour of the Z1-A120-Z2 and Z2-A120-Z2 ELP constructs at 20 uM and 5 uM concentrations measured at 600 nm. The temperature was varied with 0.5 °C per minute. The transition temperature of Z2-A120-Z2 is about 17 °C and Z1-A120-Z2 has a transition temperature of around 20 °C.

fig6

Figure 5 Temperature dependent behaviour of the Z1-A120-Z2 and Z2-A120-Z2 ELP constructs at 20 uM and 5 uM concentrations measured at 350 nm. The temperature was varied with 2 °C per minute. The transition temperature of Z2-A120-Z2 is about 17 °C and Z1-A120-Z2 has a transition temperature of around 20 °C.

fig7

Figure 6 Temperature dependent behaviour of the Z1-A120-Z2 and Z2-A120-Z2 ELP constructs at 20 uM and 5 uM concentrations measured at 600 nm. The temperature was varied with 2 °C per minute. The transition temperature of Z2-A120-Z2 is about 17 °C and Z1-A120-Z2 has a transition temperature of around 20 °C.

Dye release from hydrogel

Inhibition of bacterial growth

To follow the growth inhibition of the bacteria because of the hydrogel, a calorimetric assay was conducted with CCK-8. This type of assay can be used to detect the concentration of live bacteria in a sample and relies on the reaction between CCK-8 and dehydrogenase, which results in the formation of orange-yellow formazan. The concentration of live bacteria is proportional to the absorbance value of formazan measured at 450 nm. According to literature, the OD600 is proportional to the number of bacteria in the sample, and the relation between the OD600 and OD450 measured in samples containing CCK-8 has been shown to have a linear relationship. Based on this information, a standard curve was made that relates the OD600 to the OD450. This curve was used in further experiments to determine the number of live (and over-time thus dividing) bacteria in each sample.

fig8

Figure 7 Standard curve relating the OD600 and OD450 of bacterial samples containing CCK-8. Imaging experiments

References

[1] Despanie, J., Dhandhukia, J. P., Hamm-Alvarez, S. F., & MacKay, J. A. (2016). Elastin-like polypeptides: Therapeutic applications for an emerging class of nanomedicines. Journal of Controlled Release, 240, 93–108. https://doi.org/10.1016/j.jconrel.2015.11.010

<!Sequence and Features!>

<!REMARK: er stond eerst: , and Elastin-like polypeptide sequence A[40]I[60] BBa_K4905002. Heb daarvan gemaakt twee keer A[60]I[60], aangezien dit die met A[120] is.!>