pMK1 With Four Resilin Repeats
For the specific properties of our hydrogel, we needed to synthesize resilin consisting of 32–64 repeats to meet our requirements. A simple way to achieve this was by using the pMK1 vector, which was kindly provided by Elke Deuerling. By designing repeats flanked by two type IIS endonucleases, it is theoretically possible to generate as many repeats as needed for the intended application.
The design of our repeats (RE4) flanked by BsmBI and BsaI recognition sites was successfully completed. These restriction sites were additionally flanked by SacI and NdeI to facilitate ligation into the pMK1 vector. The pMK1 vector and the RE4 fragment, which was provided by IDT, were both digested with the appropriate restriction enzymes.
Subsequently, RE4 was successfully cloned into the pMK1 vector, resulting in the plasmid pMK1_RE4. The results were confirmed through restriction digestion (Fig. 1) and sequencing.

First Repeatigo Trial
The method utilising the pMK1 vector presents a challenge in the generation of a fragment with numerous repeats. As the number of repeats increases, the consumption of resources and time also rises. For a fragment requiring 64 repeats, the estimated cloning time using this method is at least 17 days. However, our proposed approach could theoretically generate the 64 repeats and potentially many more within a two-day timeframe.
For the method we designed 6 Oligos. First, the middle primers and also the end and start oligos were cooled down from 90 degrees to RT within 12 min. The middle oligos were then combined with the start oligos and incubated overnight with a T4 ligase. The next day, the end oligos were ranigated for one hour to obtain fragments with a maximum size of 300-500 bp (Fig. 2).

Next, we tried to extend the time in which the oligos cool down from 90 °C to 20 °C and in which they anneal to approx. 35 min. In addition, we only used half the concentration of oligos with the same volume in one sample.
As can be seen, this allowed us to create much longer oligos. We were able to show that the length of the cooling time plays a role and that a lower concentration also has a positive effect on the length of the fragments (Fig. 3).

After this, we purified several different sizes of fragments from the gel, but none of the PCRs were successful (see Fig. 4).

After this we improved several steps:

1. We optimized the gel purification by purchasing a new gel purification kit and optimizing our purification protocol by adding an additional ethanol purification step with 70% ethanol and longer ethanol evaporation phases.
2. We tried to amplify only the middle oligos overnight in three out of six samples (see Fig. 5) without adding end or start oligos (these were added the next day). Doing this, we were able to generate long oligo fragments even though the concentrations were up to 10 times higher than usually. This also helps to increase the purity of the isolated fragments.
3. We switched to a more efficient T4 ligase. Our previous ligase could ligate 0.02 pmol of insert in a 20 µL reaction, while the new ligase was capable of catalyzing 1 nmol under the same conditions. This was important as we had large quantities of DNA that had to be aligned.

Thanks to this, we finally were able to successfully amplify a fragment of approximately 1300 bp hinting to approximately 32 consecutive resilin repeats (Fig. 6).

To reach a higher concentration, which is essential for the restriction ligation of resilin and the pET28-c plasmid, we performed several PCR reactions to amplify our resilin sequence.

The restricted pET28-c and resilin sequences were effectively ligated into the pET28-c_resilin plasmid. In comparison to the negative controls of undigested pET28-c plasmid lacking the resilin insert, the plasmids containing the resilin insert demonstrate a larger size, which corresponds to an approximate size of over 1 kb. As previously demonstrated, the size of the resilin fragment, synthesised using the repeatigo method, was approximately 1.3 kb (Fig. 7).

A colony PCR was conducted to confirm the restriction ligation reaction of pET28-c and resilin, as well as the capacity of the plasmid to be transformed into bacteria. The formation and subsequent transformation of pET28-c_resilin in DH5 bacteria was successful, as evidenced by the synthesis of an approximately 1.3- 1.5 kb resilin sequence, which was achieved through the binding of the resilin-specific oligo primer to the resilin sequence incorporated in the pET28-c plasmid.

EGFP

HQSA

Hyaluronicacid