Cloning large DNA constructs as needed for synthetic biology is a major challenge. We designed a software-assisted, user-friendly and highly efficient assembly strategy. AssemblX cloning is scar-free, overlap-based and sequence independent. Using the software even unexperienced users can easily design and clone constructs with up to 25 functional units.
Without laborious parts domestication mulit-gene modules are assembled via standardized and polymerase chain reaction-free schemes in a rapid and effective process. Selection and marker switching strategies are used for best results. Final constructs can be transferred to any desired expression host.
Regulated protein expression is often dependent on chemical inducers although these are expensive and might show toxic effects on the production organism. In comparision, light is a cheap and mostly inert inducer that can be used in synthetic biology.
Our established red light system uses artificial transcription factors composed of a photoreceptor-interacting-factor protein pair, TALE-based DNA-binding domain and VP64 activation domain. Gene expression is activated via red light pulses. For the fully active and independent system we added two enzymes for cofactor biosynthesis into the yeast Saccharomyces cerevisiae.
Synthetic genetic constructs for the production of large cohorts of proteins need the option for regulated protein expression. We designed different artificial transcription factor systems that together with their cognate artificial promoters enable different kinds of regulation.
First system we designed is based on TALEs. These DNA-binding proteins can be coded to bind to the DNA sequence of interest by combining different base-specific protein domains. In combination with yeast or viral activation domains, synthetic transcription factors can be built. Similar regulation systems can be designed based on the dCas9 protein which binds to the DNA sequence of interest via a complementary guide RNA.
Second system we designed is based on plant transcription factors. Although their target DNA can not be designed, usage of protein families unique to plants allow the generation of an orthogonal regulation system in yeast Saccharomces cerevisiae.