Part:BBa_K5077019
pET28c(+) with 64 resilin repeats
The composite part was produced using the Repeatigo method. This involved creating 64 repeats of the optimised resilin exon (BBa_K5077000) and subsequently cloning them into the pET28c(+) vector (BBaK5077017), with NdeI and SacI used as restriction sites.
Plasmid for expression of long resilin threads for crosslinking
We would like to submit a nomination for our plasmid pET28c(+) with 64 resilin repeats. The plasmid can be used to express 64 repeats of resilin exons, resulting in the formation of large resilin threads. These threads can be used to create a hydrogel via crosslinking with hyaluronic acid. The resilin sequence was cloned using the Repeatigo method before inserting it into the pET28c(+) backbone.
Let us introduce you to the Repeatigo Method
We developed the Repeatigo method to generate 64 repeats of the same gene sequence. Our aim was to create super-long resilin filaments to enable cross-linking with chemically modified hyaluronic acid. Since repetitive sequences of this extend can not be synthesized, we had to develop a new method - the Repeatigo method. For this, we designed 6 different oligos. Two of them will form the main body of the insert and the other 4 will be the end oligos for both sides. One side will have an NdeI restriction recognition site incorporated and the other side will contain the sequence for SacI restriction recognition site. The idea of this method is to synthesize single stranded oligos that will assemble while repeatively creating overhangs for further assembly. Finally double-stranded DNA will be generated containing several consecutive repeats of the same sequence.
Figure 2 includes a demonstration of the method. Now let's go into more detail. In our case, we were trying to generate 64 repeats of resilin. Therefore, one resilin exon was the codon optimized sequence for the expression in E. coli. This sequence was our first oligo. We named it Oligo 1 repeat (BBa_K5077001). This oligo represents the resilin exon as a whole and is 45 bp long. The second oligo is also the resilin exon as a whole, but arranged differently. Oligo 2 repeat (BBa_K5077002) is made up of the complementary bases of oligo 1 repeat shifted by half. This means that when these two oligos bind together, they only align for 23 bp and have overhangs on each side. This allows them to grow on both sides by binding more and more Oligo 1 repeats and Oligo 2 repeats. It is important to have phosphorylated ends at the oligos to enable ligation in the end.
Then there are the end-oligos. We made four different ones. The first two are almost complementary. Oligo NdeI 1 end (BBa_K5077003) and Oligo NdeI 2 end (BBa_K5077004). Oligo NdeI 1 end is only 21 bp long and contains the NdeI restriction site (CATATG) and 15 bp of random base pairs to provide a small landing strip for the restriction enzymes. Oligo NdeI 2 end is again the complementary oligonucleotide, but contains the first half of the oligo 1 repeat as well as the restriction site and random base pairs. This results in a 43 bp fragment which ensures that the sticky ends of the middle-oligos are fully covered and blunt ends are created without gaps in the sequence.
The other end-oligos have the SacI restriction site. Again, Oligo SacI 1 end (BBa_K5077005) is only 23 bp long and contains the restriction site (GAGCTC) and the random base pairs for the restriction enzyme landing strip. Oligo SacI 2 end (BBa_K5077006) is 46 bp long and contains the second half of the oligo 2 repeat as well as the restriction site and the random base pairs. This again ensures that the sticky ends are completely covered and we get full repeats of the optimized resilin exon.
We then seperately annealed the end-oligos with NdeI, the end-oligos with SacI and the middle-oligos in three different tubes. Then one pair of oligos were annealed with the assembled middle-oligos before adding the second end-oligo pair. We then did a final ligation step to combine all fragments and checked with an agarose gel that we had produced the desired fragment lengths. The result will look like a smear because by running the assembly there statistically be various lengths of the sequence (fig. 3).
We cut out the DNA at the height of our desired fragment lengths and used it for the following PCR to amplify the DNA. This resulted in thicker bands and the fragments were at least very similar in length (fig. 4). These bands were also cut and cleaned.
The sequence was then inserted into pET28c(+). Afterwards, the plasmid was transformed into E.coli Top10 cells. We put them on LB-Kanamycin-Agar, incubated them overnight and a few colonies grew! We did a Colony PCR to verify that our insert of resilin repeats is inside the growing E.coli cells and the results were positive (Fig. 5).
We had bands at the height of 1.5 kb, which proves that we had approximately 32 repeats inside of our vector.
Hereby we confirm that the Repeatigo method works. We were able to clone 32 consecutive repeats of resilin. The Repeatigo method works as shown and can be used to create repetitive sequences for usage in the lab.
Summary
With the Repeatigo method it's possible to generate many repeats of the same gene or exon in our case. We were able to generate resilin fragments up to 3 kb in length. So, in theory, we made 64 resilin repeats, as each is 45 bp long. In the lab, we decided to try different lengths and also ligated 2 kb and 1 kb with our pET28c(+) vector after restriction. We transformed some E. coli Top10 cells with the whole vector and grew them on agar plates containing kanamycin. We also sent the plasmid for whole plasmid sequencing and got back results showing that we had created a plasmid with XX repeats of our resilin exon.
How do YOU benefit from Repeatigo?
The great thing about Repeatigo is that it is completely customizable. If you are trying to generate a lot of repeats of a single exon or gene, you have probably encountered the same problems as we have. But Repeatigo can be a solution to your problem. Simply replace the repeat sequence with the one you want. Then you can use our oligonucleotide design and fully customize it. If you want to have other restriction sites than the ones we have used, just enter your desired restriction site. You can benefit from our protocols as we have been successful in generating at least XXX repeats. If you have any questions about Repeatigo, please contact us referring to the team of 2024. We will be happy to help you achieve the same results as we have!
Next steps
As time is short, we are checking the expression of the resilin using E. coli after the wiki freeze and will present the results in Paris.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 5030
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 5153
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4964
Illegal XhoI site found at 5162 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 5030
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 5030
Illegal NgoMIV site found at 137
Illegal NgoMIV site found at 3184
Illegal NgoMIV site found at 3344
Illegal NgoMIV site found at 4932 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 2263
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