When we began designing our project, we first needed to determine the desired function of our device. Then we had to find out which DNA sequences would be able to perform the functions we had in mind. At first, we focused on the coding sequences (CDS) of our proteins of interest. Afterwards, we had to find suitable tags for the detection of our protein on Western blot, and signaling sequences that would carry our proteins through the cell membrane and cell wall of our host organism. Finally, we had to decide which promoter, ribosome binding site (RBS) and terminator we would use for the expression of our synthetic genes. In Table 1 you can find all the Basic Parts used in CYANOTRAP. If you are interested in specific features of each part, go to the Design of Basic Parts in section Specific project design or click on the part number.
Table 1. Basic Parts of CYANOTRAP.
After completing the list of our Basic Parts, we had to combine them into functional units. It was like taking single LEGO bricks of different colors and building something that makes sense. Only without the user manual.
The order of regulatory elements was clear - first comes a promoter and then an RBS. Then there is the coding sequence which ends with a STOP codon. On both ends of this sequence we added restriction sites (EcoRI at the 5' end and HindIII at the 3'end) so it can be cloned into the ectopic integration plasmids pDG3661 and pDG1664. When designing our constructs, we used codon optimization to avoid the EcoRI and HindIII sites within the coding sequence.
Our expression cassettes do not contain a terminator because Dr. Krásný advised us not to use it (based on his vast experience with gene expression in B. subtilis). We obtained one plasmid from Dr. Krásný, that already contains a terminator sequence and if the expression did not work as is, it would be possible to clone it at the 3' end of our expression cassette. It could be done through the BamHI restriction site, conveniently placed near the 3' end of the region where we want to insert our construct.
When creating the constructs, we made some mistakes at first - like putting the tags in front of the signal peptide sequence, which would have resulted in the loss of the tag. Fortunately, our PIs checked everything we prepared and made us aware of every problem. So we managed to build our own set of synthetic gene cassettes - the Composite Parts of CYANOTRAP (Table 2). If you are interested in specific features of each part, go to Design of Composite Parts in section Specific project design or click on the part number.
Table 2. Composite Parts of CYANOTRAP.