Contribution

As every iGEM team does, we're standing on the shoulder of giants. Our project would not have been possible without all the work of scientists and engineers working in the field of synthetic biology and other disciplines. Great projects are not only about achieving great results simply for the sake of it, but about giving back and contributing to the community which made it possible in the first place. This is why we are enthusiastic about our two contributions that we can make to the iGEM community this year. The first are two BioBrick parts for creating custom DART VADAR sensors. The second is a very useful software tool for creating the necessary sensor insert for it. We believe that our contributions are going to be of great value to future iGEM teams and the synthetic biology community as a whole.

ASTERISK BioBrick parts

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The key engineering challenge for our project was the creation of a plasmid that enables the in-vitro-transcription (IVT) of our ASTERISK mRNA constructs, with the ultimate goal of killing tumor cells by transfecting them with the therapeutic mRNA. Based on the published DART VADAR open-reading-frame (ORF) by Gayet et al., which is encoded on a eukaryotic expression plasmid, we re-designed the ORF for in-vitro transcription and BioBrick RFC10 conformity.¹

We put the ORF under the control of a T7 promoter and added a T7 terminator at the end, so that it could be in-vitro transcribed using T7 polymerase. In addition, we removed the original PolyA-signal, since we have chosen to add the PolyA-tail in a subsequent step with an IVT kit. Several illegal restriction enzyme recognition sites in the original ORF were silently mutated for RFC10 conformity. Perhaps one of the most useful modifications was the addition of SphI and SalI restriction sites flanking the payload sequence, making the ORF modular and allowing easy modification of the payload sequence using simple restriction cloning. We also swapped out the original TagBFP transfection marker for an mCherry coding sequence to facilitate measurements with our devices. Finally, we changed the 5' and 3' untranslated regions (UTR) to increase the stability of the mRNA.

We have developed two versions of our constructs, called ASTERISK-HBA1 and ASTERISK-HBB. The first of these (HBA1) contains 5'UTR and 3'UTR sequences of the human alpha globin 1 gene, the other (HBB) contains sequences of the human beta globin gene. These UTRs were selected because they are used in mRNA vaccines and have a strong stabilizing effect.²

Both of these constructs are made available to the iGEM community as new composite parts (BBa_K4877021, BBa_K4877022). Read more about our parts here. We cloned both constructs into the pSB1C3 plasmid, thereby generating our IVT-ready expression plasmids: pASTERISK-HBA1 and pASTERISK-HBB. The only thing missing is the 123 bp sensor sequence, which can easily be cloned into the cloning site using a HindIII digestion followed by Gibson Assembly. In this way, we have produced two plasmids that enable anyone to easily clone their desired sensor sequences into the ORF and simply replace the desired payload gene. The prepared plasmids can then be used as templates for IVT applications. In combination with our DVSensor software tool, which can generate the required 123 bp sensors for any mRNA target, we offer the community a combined toolset for rapid engineering and development of mRNA sensors based on the DART VADAR technology.

DVSensor – a tool for creating DART VADAR sensors

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As we began to outline ways to create tumor-killing mRNA sensors using the DRAT VADAR technology, the need for a new software tool emerged. Early on, we had decided which tumor-specific transcripts we would target – the EGFR and the mutant IDH1(p.R132H) transcripts – but had yet to design suitable sensors against these targets. Even if you restrict your design process to considering only the 3'UTR of these transcripts as targetable regions, you are dealing with sequence lengths on the order of kilobases. Finding suitable segments in these regions of 96 bp to use as trigger sequences for a sensor would be a cumbersome task if done by hand. You would first have to locate all suitable triplets (e.g. CCA, CAA, ...) in the region, then construct sensors targeting a 96bp segment centered on those triplets, then check for additional start- and stop-codons in the created sensors that might interfere with the mechanism, and finally check whether or not other transcripts might produce an off-target activation due to sequence similarity to the target.

It is obvious that it is virtually impossible to generate all possible sensors for a given target transcript by hand in a reasonable amount of time. Thus, we have created DVSensor – a software tool that automates the entire task and provides an easy-to-use graphical interface for controlling the process. The software allows you to generate all possible sensors for any mRNA target of your choice, and even checks the specificity of the generated sensors using BLAST database searches to identify other transcripts that could cause off-target activation. It accepts target mRNA sequences in the standardized FASTA or GenBank record formats as input and produces a table in CSV format with all results. Read more about our tool on our software page.

We anticipate that the novel DART VADAR technology – similar to toehold RNA switches in the past – will be used in many future iGEM projects and are proud to provide the community with the first tool of its kind for creating your own sensors tailored to your specific needs. We believe that our software contribution will save future iGEM teams a lot of time and effort during their engineering design steps and enable them to design the right mRNA sensors for their projects.