Part:BBa_K5109001:Design

The part does not present a terminator: due to difficulties we encountered in phase of synthesis, the derminator could not be inserted into the expression cassette. Our solution was to use a specific backbone that encodes for a terminator right after the Biobrick suffix: this way, when cloned via RFC 10, the sequence will present a terminator only a few bases after the stop codon. In alternative, if the insertion of a terminator is needed, we provided two BsaI restriction sites, that can be used to insert a terminator via Golden Gate cloning. The two BsaI cutting sites make up a new basic part that we listed as:BBa_K5109002.

We choose to insert NdeI site upstream the coding sequence and BamHI site downstrem the sequence, id order to allow the interchange of different enzymes:NdeI cutting site is located upstream the coding sequence of the Dehalogenase, while BamHI cutting site is located downstream the coding sequence. When a different enzyme is used to replace the dehalogenase, the enzyme of interest has to be either synthetized with NdeI and BamHI sites at the ends, or amplified with tailed primers that could insert those sites. Then, a directed cloning can be done, via restriction with NdeI and BamHI and subsequent ligation. NdeI cutting site is also specifically designed to be used to insert a coding sequence upstream the enzyme sequence: in our case, this is necessary to insert the sequence of a membrane proteine, building a surface expression tool.