Software
As described in contributions, during the development of our project we encountered several difficulties in the process of designing in silico RNA toehold switches, mainly because there were several bioinformatic tools and the process from passing information from one tool to the next became time consuming and difficult for persons with no coding experience. Therefore we utilized the Design→Build→Test→Learn Cycle to develop an all-in-one software tool capable of designing and analyzing both primers and toehold switches.
But once we developed the first version of our software (Toehold Switch Creator) and started testing it on different operating systems, we found out that the overall installation process was tedious and difficult for Windows users. Therefore we started the cycle again and we came up with the idea of developing a web application of the software so that users could easily design their sequences without the need of installing several bioinformatic packages. And currently, we are in the building and testing process of the web app with different cloud services.
Wet Lab
Our original plan consisted of testing our toehold switches in a commercial cell-free protein expression system, however, as noted in notebook it did not work out. We needed an alternate plan so we came back to the experiment design phase of the project and set up from scratch a completely different way to test and characterize our toeholds, we decided to do it in bacteria. In the middle of the laboratory work phase we designed primers with needed restriction sites overhangs, we successfully added those sites to our constructs and finally cloned them into a set of plasmids useful to do a simultaneous transformation (cotransformation).
Our secondary plan of performing a cotransformation did not go as planned, we only got to perform single plasmid transformation successfully so we came with the idea of growing these single plasmid containing bacteria and make them competent so we could do a serial transformation adding the second plasmid afterwards which we finally were able to do.