Innovative BioBrick construction
This year we developed an innovative way to construct and pre-confirm BioBricks efficiently.
This method mainly applies to those parts that are initially constructed on backbones other than pSB1A3/1C3/1K3 etc. and suffer adding suffix/prefix when changing into standard backbone.
Due to various and specific needs for experiments, or customs in host labs, it is often unfeasible to use the standard backbone from the beginning to the end of the project. Therefore, when we construct BioBricks, we will have to reconstruct the part into the standard backbone.
For example, this year, we used pEntry for most of our experiments, which contains attL recombining sites required by our design. We also used eukaryotic vectors for cell experiments and our G1 promoters (intended to submit as the new basic parts) were are ligated in an eukaryotic vector.
Moreover, sometimes it is impossible to construct the parts with prefix/suffix for experiments: there is a big chance that the backbones used for experiments contain PstI, EcoRI, NotI enzyme sites (one or more), and that it will be used for experimental needs. In this occasion, you don’t dare add the same enzyme site beside you parts!
Thus while we suffered from reconstructing parts for standard BioBricks, we eventually developed this new method. Since this is a common issue in iGEM experiments, its significance can be easily seen.
Method and protocols
- Add XbaI at the 5’ region of the part and SpeI at the 3’ region by PCR.
Advantage: both prefix and suffix have 22bp in length, which is unfriendly for PCR: the matching region of the primer with the part should be no shorter than the unmatching tail, requiring at least 44bp in total. The Tm of the primer will be huge, especially when the parts’ matching region is also unfriendly. Thus it will be more feasible and efficient to add a 6bp tail rather than a 22bp tail.
- Digest pSB1C3 and the PCR product with XbaI and SpeI enzyme. Follow the instruction of the enzyme protocol.
- Treat the digested pSB1C3 with alkaline phosphatase, which can remove the phosphate group of the cohesive ends of the backbone. Follow the instruction of the enzyme protocol.
Explanation: As XbaI and SpeI are isocaudarners, this step is necessary to prevent the backbone from self-cyclizing without the part. Treat with alkaline phosphatase every time before use.(Optional: treat the digested part with polynucleotide kinase, assuring that the ends of the part have phosphate groups. Follow the instruction of the enzyme protocol. )
- Ligate the digested part and the digested-treated backbone, and transform into E.coli.
- Product confirmation: After plasmid extraction, use enzyme XbaI digest the plasmid, of use Xbal and SpeI to digest the plasmid.
Explanation: XbaI and SpeI are isocaudarners, so theoretically there is a 5 to 5 chance that the part is ligated head-to-feet in the backbone. Luckily, neither can XbaI or SpeI digest the wrong ligated plasmid. Therefore one enzyme digestion can tell you are right or wrong. (Xbal is cheaper than SpeI, so choose XbaI). If you also want to confirm the size of your part, use both Xbal and SpeI.
- Electrophorese the digestion product, and confirm your plasmid.
We used this method to construct our BBa_K1926011, BBa_K1926012, BBa_K1926013,BBa_K1926002 and BBa_K1926003. We successfully obtained the right plasmid after the first round of molecular clone.
We also tried the method on BBa_K1926021 and BBa_K1926022 (the size of which twice as big as the former), but other than one head-to-feet product, we didn’t obtain the right plasmids.
Therefore we suggest that this method is mainly suitable for parts smaller than 1kb, as the longer the parts, the bigger possibility for it to self-cyclize, which wastes the ligation substrates.