BBa_K2002000 is a composite part that is composed of the CDS of the LJM11 saliva protein gene that has been codon-optimised for bacterial expression. Upstream of this gene is a His tag and a secretion signal USP45 that has been demonstrated to work as a secretion signal from previous literature . A RBS was also included of this to allow for efficient translation of the mRNA produced following transcription.
Downstream of the LJM11 CDS is a terminator (BBa B0011) which allows for Rho-independent termination of transcription.
The biobrick prefix and suffix is included on the 5’ and 3’ ends of the molecule respectively. Biobrick incompatible restriction sites were removed by codon-optimisation.
LJM11, as described on the project page, encodes for a saliva protein found in a sand-fly species and has been can induce an immune response.
BBa_K2002001 is a device that can be used as a reporter system in in vivo or in vitro models. Our experiments have shown that it can be used in in vitro experiments but equally it could be used in in vivo models. The part contains an iRFP CDS. A outlined in the project section, this is an infrared fluorescent protein that will only fluoresce in mammalian cells or tissue.
BBa_K2002001 contains a RBS for efficient protein translation, a his tag to allow our protein to be purified via a nickel column and the BBa_B0011 terminator to stop the transcription of the mRNA in the cell.
As described in the iRFP section of the project. Bacterial expression of iRFP can be shown to work if the media is supplemented with FBS and an in vitro validation using RAW 264.7 macrophages was also carried out to demonstrate the functionality of our concept in a real world setting.