Part:BBa_K4905006

Elastin-Like Polypeptide Triblock with Leucine Zippers

This part is made up of parts BBa_K4905004, BBa_K4905005, BBa_K4905001 and BBa_K4905002 . It was used by the 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 leucine zippers that have affinity for each other. ELPs have hydrophilic and hydrophobic domains that exhibit reversible phase separation behavior that is temperature dependent. This allows them to be used in the formation of a reversible hydrogel. Protein expression and purification The protein has an expected molecular weight of 105.1 kDa Organic solvent extraction, ITC SDS-page

13. Z1-A120-Z2 14. Z2-A120-Z2 15. 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.

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.

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.

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.

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.

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.

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

Sequence and Features

Assembly Compatibility:

• 10
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• 12
  INCOMPATIBLE WITH RFC[12]

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• 21
  INCOMPATIBLE WITH RFC[21]

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• 23
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• 25
  INCOMPATIBLE WITH RFC[25]

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• 1000
  COMPATIBLE WITH RFC[1000]