Dry Lab
Our Dry Lab work revolves around models that were developed in the Salis Lab before our time here. These models can be accessed through De Novo DNA.
The Riboswitch Calculator - Design Mode
The most important inputs for the Riboswitch Calculator include the aptamer sequence, aptamer structure, promoter sequence, CDS sequence (sequence of the downstream gene), ligand-apatamer binding free energy. Using information from Aptagen’s Apta-Index, we were able to gather the aptamer information and identify apatamers that would be relevant to diagnostics. After a few minutes, we sent in the design job. The algorithm produced results within minutes, which include pre-aptamer and post-aptamer sequences that work with the aptamer region to regulate translation and form the entire riboswitch. For example, the Riboswitch Calculator produced 153 riboswitch sequence predictions for bFGF.
This process was repeated for the each of our six aptamer sequences. We sorted the results by their activation ratios and selected five sequences with high activation ratios and varying ON states. This would allow us to examine a wider range of ON states and would possibly avoid the problem of all riboswitches being too sensitive or not sensitive enough.
More details about using the riboswitch calculator and interpreting results can be found on our Contribution page.
The Riboswitch Calculator - Predict Mode
Predict Mode can be used to predict the function of an existing riboswitch sequence. The inputs for Predict Mode are similar to Design Mode, except it will require known pre-aptamer and post-aptamer sequences. The outputs include predicted structural changes, translation rates, switching free energies, and more. Predict mode became especially useful to us when we found mutations in our designs after sequencing. Specifically, our pFTV1_T7_bFGF2_mRFP1 plasmid had a single insertion in the post-aptamer region, and we wanted to test if the riboswitch would still be viable. After plugging it into Predict Mode, we found that it still had a sufficient activation ratio, ON state, and switching free energy.
CDS Calculator
The CDS Calculator inputs coding sequences and codon optimizes these sequences for your organism of choice. In our case, we needed to design a CDS for mRFP1. Some functions of the CDS Calculator include avoiding including certain digestion cut sites, avoiding introducing promoter motifs, and increasing stability. We then used our codon-optimized CDS to design our riboswitches.
Context Aware Autoalign
Context Aware AutoAlign is a useful tool for sequence confirming DNA in bulk. With over 60 unique plasmids, we became very familiar with sequencing. With Context Aware AutoAlign, we could easily upload a list of reference sequences and all the files from our sequencing results to find how well they matched. The tool is quick and useful, and it especially helped us when some mistake mixed up our sequences and mislabeled our alignments!
