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 (see Fig. 1) and sequencing.
Fig. 1: 1 % agarose gel after double digest of pMK1 and pMK1_R4 with SacI and BsaI. Lane 2 and 5 with digested pMK1 shows two bands at around 3000 bp and 600 bp. Lane 3-5 and 6-9 with digested pMK1_R4 show two bands each at around 3000 bp and 200 bp.
Additionally the insertion was confirmed by Sanger sequencing (see Fig. 2). The sequencing also showed that there where no mutionas in the RE4 sequence.
Fig. 2: Sequencing result of the RE4 region in the pMK1_RE4 Plasmid. On A is shown the first halve of the RE4 insertion and on B is shown the second half of the RE4 insertion.
Repeatigo Method
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 (see Fig. 3).
Fig. 3: The first trial of the Repeatigo method to produce repeat sequences of resilin. Lane 2-4 show results of different ratios of start- and middle-oligos. The gel shows smear bands in every sample lane centering around 100 bp. 1 % agarose gel.
Fig. 4 The second trial of the Repeatigo method to produce repeat sequences of resilin. Lane 2 and 3 show results of the ligation using either 4 µg (lane 2) or 2 µg (lane 3) of middle-oligos for the ligation. 1 % agarose gel.
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. 4).
After this, we purified several different sizes of fragments from the gel, but none of the PCRs were successful (see Fig. 5).
After this we improved several steps:
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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.
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We tried to amplify only the middle oligos overnight in three out of six samples (see Fig. 6) 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.
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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.
Fig. 5: Gradient PCR to verify the correct annealing temperature for the oligos. Ladder: 1 kb Plus; agarose gel (1 %).
Fig. 6: Oligo Fragments of different lengths. Different concentrations of middle-oligos (see above the lanes) resulted in different length distributions of resilin fragments. Three different fragment lengths were isolated from the gel (see red boxes); 1% agarose gel; DNA Ladder: 1 kb Plus.
Thanks to this, we finally were able to successfully amplify a fragment of approximately 1300 bp hinting to approximately 32 consecutive resilin repeats (Fig. 7).
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.
Fig. 7: PCR for amplification of resiilin oligos with a lenght of about 1,3 kb from two different resilin reactions. 1% agarose gel; DNA Ladder: 1 kb Plus.
Restriction Ligation of pET28-c with resilin
Fig.8: pET28-c_Resilin PCR. pET28-c not digested (negative control) and digested *pET28-c 1-3 triplicates as well as digested *pET28-c_resilin. Additionally, the resilin sequence, as well as the undigested pET28-c_resilin 1-3 are shown. Ladder: 1 kb Plus; agarsoe gel: 1%
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.
Colony PCR of TOP10 transformed with pET28-c_resilin
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 TOP10 and DH5-α 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.
Fig. 9: pET28-c_Resilin colony PCR. Four reactions were made with different clones from a transformation of DH5a with the pET28-c_resilin. As a positive control the untransformed pET28-c_resilin was used and as a negative control water was used as a template. Ladder: 1 kb Plus; agarose gel: 1%.
Sequencing
In the Next step the colonies where sequenced by Sanger sequencing. Here the isnertion of the 1.3-1.5 resilin sequence into the pET28-c plasmid could not be confimred (See Fig. 10). As one can see, the sequenzing results could only be alligned to the vector pET28-c but not to the insertet repeats.
Fig. 10: Sequencing result of on of the colonies. On the lower figure the filled red line indicaeds the sequenced sequence matches the desired sequence. Where the red line is not filled, the sequenced sequence does not match the desired sequence. In the upper figures, the sequencing of both ends of the RE32 insertion was zoomed in on
Detection of Resilin Expression
Following the successful transformation of the pET28-c_resilin plasmid into competent JM109 bacteria of the novel reCA-deficient expression strain, the expression of resilin was induced with IPTG. After 21 hours, the cells were either lysed with only SDS-sample buffer (Figure 11), sonification (Figure 12) or heat (Figure 13), and subsequently analysed by SDS-PAGE.
Fig. 11: SDS page before (b.i.) and after (a.i.) induction of E. coli strain JM109 with IPTG for resilin expression. The bacteria were transformed with the pET28c(+) plasmid with (R) and without ((pET28c(+))) our resilin insert. Two different resilin samples were used. Resilin 1,2,3 was used three times and resilin 2 twice. Samples were mixed 1:1 with SDS loading buffer. 10% gel, ladder: PageRuler unstained protein ladder.
Fig. 12: SDS page after induction of E. coli strain JM109 with IPTG to detect resilin expression. The bacteria were transformed with the pET28c(+) plasmid with (R) and without (pET28c(+)) our resilin insert and two different resilin samples were used. The resilin 1,2,3 was used three times and the resilin 2 was used twice. Lysis was performed by sonication and each sample was lysed once in LB medium and once in lysis buffer (lysis). 10% gel, ladder: PageRuler unstained protein ladder.
Fig. 13: SDS page after induction of E. coli strain JM109 with IPTG to detect resilin expression. The bacteria were transformed with the pET28c(+) plasmid with (R) and without (pET28c(+)) our resilin insert. Two different resilin samples were used. Resilin 1,2,3 was used three times and Resilin 2 twice. Lysis was performed by heating the cells at 95°C and each sample was lysed once in LB medium and once in lysis buffer (lysis). 10% gel, ladder: PageRuler unstained protein ladder.
The expressed resilin protein of the 30 resilin repeat sequence should exibit a molecular weight of approximately 50 kDa. Unfortunately, no bands at about 50 kDa were visible compared to the respective controls (Figures 11-13). To conclude, further research is required to repeat and optimise the expression of resilin.
