With our MocloMania parts collection, we want to facilitate the genetic assembly and expression of fusion proteins in Leishmania tarentolae. By establishing parts that are suitable for Modular Cloning, assembly of genetic constructs can be realized as easy and efficient as possible. With the help of MoClo, target protein sequences can be freely assembled from a variety of L0 basic parts into a cohesive L1 genetic construct. For our MocloMania system, this assembly includes the insertion of the target sequence into weird_plex, our L1 expression vector. Our goal was to test the quality of this expression system by producing, detecting and purifying recombinant SARS-CoV-2 receptor binding domain (RBD) in Leishmania.
Regarding the research aim of our project, it doesn’t come as a big surprise that the majority of our wet lab time was spent cloning different L1 constructs and transfecting them into Leishmania cell culture. We ended up creating almost 20 different constructs with variable purification or detection tags. Most of them involved the SARS-CoV-2 receptor binding domain in variable cloning positions as well as our sAP secretion signal to mediate protein secretion into the culture medium. We of course tried to incorporate every single one of our basic parts into a composite construct, not only to confirm its correct adaptation towards the MoClo cloning standard, but also to verify the functionality of its protein counterpart.
While all of our basic parts could be successfully introduced into L1 constructs and mostly showed decent transgenic expression levels on western blot, we struggled with optimizing the actual downstream purification and detection procedures. In order to find out more about our over-all experimental success, please refer to our Results page. For more detailed information on a specific L1 construct and its experimental outcome, please feel free to check out its registry page on the iGEM Parts Registry. We have listed all the L1 constructs assembled during our project work in the table below, organized by expression localisation as well as cloning position of the SARS-CoV-2 receptor binding domain sequence.
