We believe that our team has made the following contributions to the iGEM community.
Design of Positive Control DNA Fragment
Many iGEM teams, including us, rely on external sponsorships and funds from the university. We understand that most teams are very short on funds and, thus, cannot replace expired items at will. Our positive control fragments allow teams to assay the functionality and activity of the restriction enzymes, saving money from using them to replace expired but functional enzymes and saving time from just hoping that the expired enzymes would work. The unique property of the positive control fragment is that it has many restriction sites in a short sequence, resulting in the effective use of dNTPs for building DNA fragments.
Use of Nickase for Producing Short ssDNA Fragments from RCA
In our original design, we planned to enhance the production speed of G-quadruplex DNAzyme (G-quad) and, therefore, colour change by cleaving the primer-G-quad phosphodiester bond on the product strand, freeing the DNAzyme and the primer for more reactions. However, the attempt with 13PD1 DNAzyme failed for us (see DNA-Cleaving DNAzymes Test). This has forced us to think innovatively, and we turned to the enzymes currently in the reaction mix, nickase. By leveraging the ability of nickase to cleave only one strand, we can achieve amplification of RCA initiation, hopefully producing faster results for our kit.
Development of Robust ssDNA Circularisation Protocol
At the heart of our project lies RCA, which directly controls the production of G-quad, leading to visible colour change. RCA relies on circular ssDNA templates, which are very expensive if they were to be synthesised by companies. Our team also could not afford to synthesise circular ssDNA, so we turned to the literature and found a protocol for ssDNA circularisation with limited success. We encountered false positives when performing RCA due to the residual scaffold hybridised to the circularised template, acting as a perfect primer for RCA. We then modified the protocol with the use of exonuclease III to also remove the circularised RCA template with RCA scaffold hybridised to it. With exonuclease III, we have not re-encountered the problem of false positives. Hence, we believe this modified circularisation is robust, allowing future iGEM teams to produce circular ssDNA for their experiments at a low cost.
Parametrisation of Haem Residue in AlphaFold 3 for GROMACS Simulations
During modelling, our most prominent difficulty was the
parametrisation of haem residue for GROMACS simulations. Since
the haem residue contains iron (III) ions, which were
not present in ordinary force fields, they provided significant
hurdles during parametrisation, as
quantum calculations are necessary. Installing
quantum chemistry packages is time-consuming, and the
calculations required to obtain the parameters are lengthy and
easily consume days. In light of the problems we have encountered along the way,
and considering the popularity of the AlphaFold 3 model, we decided to share the
haem topology model with the synthetic biology community,
allowing future teams to
perform simulations containing the haem residue quickly. The
.itp topology file is available for download
here.