We added four basic parts to the registry: the ion channels mHCN2, hERG, Kir2.1, and α1G. We used these ion channels to build our project and to validate our model, but other teams might find them useful, too!
A detailed description of the biobricks created for the project can be found here
But what do we think about our biobricks ourselves? Here's our top 4:
Our favourite biobrick is the inwardly rectifying potassium channel, Kir2.1. This protein is a mammalian ion channel, and has some interesting electrophysiological properties. For example, (over)expression of Kir2.1 in HEK cells leads to differences in membrane potential.
In our project, we used this protein to verify the added value of our model. According to this model, the oscillating system could result from a combination of the mHCN2, hERG and α1G ion channels. We tested the modelled system in HEK cells, and obtained the oscillating system! However, the model1 suggested that the specific characteristics of the ion channels in the system are crucial for the system to work. Therefore, we were curious whether the oscillations would result from just any combination of polarising, depolarising and hyperpolarization-activated channels, and we tested a system consisting of mHCN2, Kir2.1 and α1G. As expected, this combination did not result in any oscillations, validating the importance of our model.
We tested the system containing mHCN2, Kir2.1 and α1G with two Kir2.1 variants: The wildtype gene, but also the carefully designed biobrick-compatible variant we have presented to the registry. This biobrick-compatible variant was then cloned into the standard pSB1C3 vector, verified using sequencing, and sent to the registry. Anyone interested in accessing the raw sequencing files can contact us by email (email@example.com) or social media. A picture of the blast of the sequencing results can be found on the registry page, as well as here.
We hope future teams will find this biobrick as as useful as we did, and look forward to seeing its future uses!
Our first runner up is the human Ether-à-go-go-Related Gene. This is not only because of its funny name: The hERG protein has been vital for building our oscillating system. Moreover, this protein has a lot of potential for future projects: Inhibition of hERG can lead to severe side-effects, and therefore it is a major antitarget in drug development. This biobrick could therefore be useful for any project related to pharmaceuticals.
During our project, we have used the wildtype hERG variant. However, we designed a biobrick compatible variant by removing illegal restriction sites and by lowering the GC content to allow synthesis as gBlock gene fragments. We have cloned this biobrick compatible variant in the pSB1C3 vector and provided it to the registry. However, due to issues caused by the size of this gene, and due to time constraints, we were not able to verify it using sequencing. However, we verified it by a restriction digestion and PCR.
mHCN is a member of the hyperpolarization-activated cyclic nucleotide–gated (HCN) family of ion channels. Their peculiar behaviour causes oscillations in both the heart and in the HEKcite system.
We have designed a biobrick compatible version of the mHCN2 gene, by removing the illegal restriction sites and lowering the GC content. However, IDT has been unable to synthesise this gene, as long, GC-rich repeats remained in the sequence. Nonetheless, we have added the sequence to the registry, as it is already possible to order this biobrick as an artificial gene. However, the DNA synthesis techniques are constantly improving, and we expect that this biobrick can be synthesised as a gBlock in the future.
The α1G calcium channel was vital in our project, in order to create the oscillating system. Furthermore, its functionality as a calcium channel allowed us to visualise a rhythm using calcium imaging in one of our earliest experiments.
We have designed a biobrick compatible version of the α1G gene, by removing the illegal restriction sites and lowering the GC content. However, the large size of this gene is not compatible with the standard registry vector, pSB1C3. Therefore, we were not able to provide a sample of this biobrick to the registry. However, it is possible to order this gene as several gBlock fragments, and assemble them by for example Gibson assembly.
References: 1.Cho, H. C., & Marbán, E. (2010). Biological therapies for cardiac arrhythmias. Circulation research, 106(4), 674-685. Chicago