Contribution

Swedish iGEM Conference

We started what we call a sister event of the ongoing Nordic iGEM Conference (NiC), which is an annual meetup of all the iGEM teams in the Nordic region, to share ideas and have a rehearsal of the final presentation before the Giant Jamboree. We had 4 iGEM teams from Sweden that had participated in the conference, namely Stockholm, Lund, Chalmers-Gothenburg and Uppsala. We thought that having workshops and a meetup before we all had started our lab work would be a great initiative to set the mood for the whole summer for the Swedish teams. We thought this will help the teams plan their projects better after early feedback from experts. We had Mr. Pontus Christoffersen, who is an expert in presentation strategies, giving us valuable advice which we could use to increase the impact of our project presentations. The 3-day conference also included a lot of fun and adventurous activities to increase the bond between the participating teams. We plan to keep this tradition alive with each Swedish team organizing this conference on a rolling basis.

Biobrick development

In many ways the gastric intrinsic factor biobricks submitted by the iGEM Stockholm 2022 team represents a spiritual successor to the work performed by the iGEM Istanbul ITU 2013 team, which also centered their project on the characterization of the same protein. It should be noted, however, that we used different restriction sites and objectives. Our goals with the project of course went beyond the characterization of the protein (the extent of the 2013 project), and thus our parts feature some notable differences/developments compared to the Istanbul team’s part. Firstly, our parts feature a LacI promoter, allowing inducibility and increasing expression. Second, and more relevant to our application, our parts feature his6 purification tags instead of the GST purification tag used by the 2013 team. This choice was made because a small tag (840.8 Da) was desirable over a larger one like GST (26 kDa) in order to minimize the risk of the tag impacting the folding of the IF (and thus, crucial epitope structure and vitamin B12 affinity for our project). We cloned our gene into a mammalian surface display vector, allowing it to be transfected into HEK cells for further study. This surface display feature is not a part of the submitted biobricks themselves, but, was integrated around them. Lastly, one of our parts (BBa_K4214004), features a periplasmic secretion sequence (pelB), yielded to a simplified purification of the protein during initial tests in our project. Together, these modifications should allow for simplified production and purification of an IF protein closer to the wild type, compared to the predecessor from Istanbul [1][2][3].

Software

In order to perform the mutagenesis of IF in a systematic manner, our drylab team created a software that allows you to input a sequence of DNA or amino acid and simulate amino acid substitutions on that sequence. The software takes various user input to optimise the mutagenesis to their specifications, such as whether to disrupt or preserve the protein structure according to a substitution matrix, how many substitutions the software should induce, and where in the sequence they’re wanted. The software also has a function to predict how the protein will be affected by a mutation, including a scoring function so it can evaluate whether the mutation will be beneficial to the protein. More in-depth information about how the software functions and about its inner workings can be found in the software documentation:

References