Week 21

We transformed the constructs from last week into Top10 competent cells. From the agar plates, we selected several colonies and grew these overnight, after which we purified the plasmid with a miniprep. To confirm that the ligation was done correctly, we did a double digestion using restriction sites just outside of our gene of interest. The results can be seen in the following image.

The ordered gBlocks had also arrived, and we digested these along with a pET24(+) vector. The gBlocks were then ligated into the empty vector.

Week 23

From each plate, we picked (up to) three colonies of which we made small cultures and performed colony PCR. The protocol for this can be found on our Experiments Page. With the colony PCR, we could confirm the presence of our desired gBlock within the plasmids. We then purified the plasmids containing gBLocks and continued digesting them, along with the plasmids containing the ELPs. These digested fragments were then ligated to obtain the following constructs:

- Z1- I_[60]-A_[100]-I_[60]-Z2

- Z2- I_[60]-A_[100]-I_[60]-Z2

- Z1- I_[60]-A_[120]-I_[60]-Z2

- Z2- I_[60]-A_[120]-I_[60]-Z2

The plasmids were transformed into Top10 cells, colonies were picked and grown into small cultures over the weekend.

Week 25

We had some trouble cloning this week since our digestions did not seem to work properly. We found out that multiple aliquots of the BglI restriction enzyme were not working. The freezer in which they were stored was set at -30°C, which led us to believe it might negatively affect the enzyme’s efficiency since the recommended storing temperature is -20°C. Luckily, one of our supervisors had a stock of BglI available stored at the right temperature. When using this stock, the enzyme cut perfectly.

Since our first zipper-containing constructs were cloned successfully, we started expressing the proteins by first transforming the plasmids into BL21(DE3). Then, small cultures were made and when the OD600 reached 0.6, they were transferred to large cultures. To check if the right proteins were expressed, we took samples at several time points and ran the cell lysate over an SDS-PAGE gel.

After 36h of protein expression, the cultures were centrifuged, and the bacterial pellets were stored at -20°C.

Week 27

With our purified ELPs, we were finally able to test their ability to form a hydrogel. Since the yield of our protein was quite small, we decided to first attempt to make a 10 w/v% gel. After dissolving the ELPs in MilliQ water at 4°C, we observed that the fluid became quite viscous, but it remained transparent. Upon bringing it to RT, it became very turbid and upon closer inspection, we saw that the gel was formed. An image of the gel in a small container can be seen in the image below.

Week 29

ELP solutions were made at low concentrations so that we could determine the transition temperatures of our ELPs using UV-vis. The results can be read on our Results Page. In addition, we upscaled our protein expression, so that we obtained some more ELPs to work with.

Week 31

To determine if we can control the division of our cells and if they are still metabolically active, we tested them using a colony-forming assay and an assay in which a substrate, Cell Counting Kit 8 (CCK8), is converted in living cells. More information about this experiment can be found on the Experiments Page and the Results Page. We compared cells in which protein expression was induced versus where no expression was induced and used ampicillin as a selection marker. The absorbance gave us an indication of how many cells were still alive and able to convert CCK8. In addition, we plated these bacteria onto LB-agar plates to see if they still managed to grow.

Week 33

We wanted to test the different diffusion rates of a fluorescent dye when this was incorporated into the gels. Rhodamine B was used initially to test the diffusion rate. More about this can be read on our Results Page. However, when attempting to form the gel, it did not dissolve very well in the water containing rhodamine B. We figured this might be due to salt left over from doing ITC purification. Therefore, we tried purifying the ELPs by dialysis again to remove the salt.

After several failed attempts at cloning the I_[60]-mNeonGreen gene into the pBAD vector, we tried a different strategy. We used BamHI-HF instead of BamHI, and we performed two single digestions, instead of one double digestion. This resulted in successful transformations. However, when we got the sequencing results, we found that the ELP sequences attached to mNeonGreen were not I_[60], but A_[3]G_[2][12], therefore the protein is from here on reffered to as such.

Week 35

Rhodamine B diffusion was also done for the 5% gel, where the concentration in the supernatant was measured every 2 hours. We also received our IL-10 gBlocks, which we first cloned into pET24a(+), as a back-up for when expression in the pBAD vector would not work.

Week 37

We managed to co-express the Z1-A120-Z2 together with A_[3]G_[2][12]-mNeonGreen. We first let Z1-A120-Z2 express in AIM TB medium under the same conditions as for single expression. Then, we diluted the culture to OD600 = 0.6 in AIM TB medium containing arabinose (6.6 mM). This resulted in successful expression of A_[3]G_[2][12]-mNeonGreen as well.

We also performed new microscopy experiments, using a viability staining called Live-or-DyeTM. Using this dye, in combination with a Hoechst staining, we wanted to see if our cells would stay alive after being treated with ampicillin. However, since the results of this experiment were not conclusive, the data is not shown on the results page.

IL-10 expression was induced using the same conditions as for A_[3]G_[2][12]-mNeonGreen. Cultures were grown to OD₆₀₀= 0.6 in TB medium, when arabinose was added to a final concentration of 6.6 mM following incubation at 37°C for 2h. Then, IL-10 was extracted using periplasmic extraction. This was done, because the native protein contains disulfide bridges, which can only be formed in the oxidizing environment of the periplasm. 10 mL of extraction buffer (0.2 M TRIS pH 8.0, 0.5 mM EDTA, 20 w/v% sucrose, 0.1 mM PMSF, Complete Protease Inhibitor Cocktail) was added per 1 g of wet cell pellet and incubated for 30 min 4°C. Cells were pelleted by centrifugation at 12.000 rpm for 10 min at 4°C, after which the supernatant, containg IL-10, was collected.

Week 39

We did the CCK8 assay for the freeze-dried samples and we also tested the gelated bacteria for resistance under oxidative conditions by incubating them with different concentrations of hydrogen peroxide. The viability of the bacteria was then also tested using the CCK8 assay.

As a final experiment, we co-expressed Z1-A120-Z2 with A_[3]G_[2][12]-mNeonGreen and used these samples to do Fluorescence recovery after photobleaching (FRAP) on. This gave us an insight into the diffusion of the fluorescent molecule through the bacteria. The results from the experiment can be seen on our Results Page.