Team:Heidelberg/Engineering
Engineering Success
Design, Build, Test, Learn...
Introduction
iGEM is an engineering competition, about developing functional biological building blocks. The process of creating such building blocks is described by the iGEM engineering cycle, which starts with carefully designing a part, then building and testing it, learning from the results and finally improving the part with the gained knowledge. Because of Covid-19 it was quite challenging especially for our wetlab projects, to advance to the building and testing segment of the cycle. Nevertheless we made an effort to follow the principles, intensively researched literature and consulted experts to create well outlined plans for our experiments:Detailed plans of our work in the wetlab
On unexpected results
The objective of science is to explore the unknown. Therefore it is impossible to predict all the possible results and solutions for them. Our strategy was to research carefully and use the available software tools like CONCORDE or the ViennaRNA webserver RNAfold, to increase the probability of our experiments functioning properly. In some cases we developed new software tools to address our problems. In case of unexpected errors we firstly planned to examine all the possible error sources in our experiments. We tried to design our experiments in the way that we could make sure that every element of the construct is working properly. If we wouldn’t be able to find the reason for an unexpected result, we would try to research the literature further or ask for advice from an expert.Examples for implemented engineering cycles in our experiments
The Broccoli Aptamer
We wanted to create an RNA binding protein with endonuclease activity, since it would expand the abilities of cellular regulation on a transcriptional level. After rigorous research we decided to execute an experiment with a complex consisting of a Pentatricopeptide Repeat Protein (PPR) and a Ribonuclease A. The first problem we encountered while designing the experiment was that we could not use a mRNA translation inhibition assay like in the (more information on the design of mRNA translation inhibition), since the PPR could have an inhibitory effect on translation of the mRNA, the fluorescence loss could be contributed solely to the complexation of the PPR and mRNA and not necessarily to the degradation of the mRNA through the ribonuclease. We found the solution in using a broccoli aptamer, which is an RNA with stable secondary structure. Broccoli can bind the fluorogen DFHBI and therefore its presence can be detected by fluorescence. Because fluorescence is not induced through translation with the broccoli aptamer, we theorized that a loss of fluorescence would indeed only be associated with the degradation of the aptamer through the ribonuclease. But beware, the use of Broccoli came with new challenges! Firstly, PPRs can bind only single stranded RNA, but Broccoli has a very stable secondary structure and has no suitable binding site for a PPR hexamer. Secondly, the original broccoli has a BsmbI cutting site, but we had to use BsmbI for the Marburg Collection cloning. These obstacles were resolved by redesigning the Broccoli aptamer computationally with a software we designed called Designsimple. If we had enough time in the lab we would firstly try to collect evidence that the redesigned Broccoli aptamer functions. If that would not be the case, we would analyse what are the other possible ways to redesign the original Broccoli. Afterwards we would continue with our experiment.
RNA*DNA-DNA triple helices
RNA*DNA-DNA triple helix have been known for a longer period of time. Recently, Kunkler et al. characterised binding energies of single RNA-DNA nucleotide pairs in the triple helix in vitro.
Another engineering question in the experiments with the triple helix was, which distance between the promoter and the binding site of the RNA should we choose. It was shown that an analogical construct using a dCas instead of a triple helix works optimally if the binding site of the guide RNA is 91 base pairs in front of the promoter.