Team:Guelph/Demonstrate
Our team designed and created assemblies of CLE18 CRISPRa & ICE2 CRISPRa with various gRNA’s
BBa_K4058018,
BBa_K4058019,
BBa_K4058020,
BBa_K4058021,
BBa_K4058022,
BBa_K4058023,
BBa_K4058024,
BBa_K4058025,
BBa_K4058026,
BBa_K4058027
These constructs were transformed from E. coli to A. tumefaciens and finally our end target of A. thaliana. This was done with the intention of increasing the expression of each of our target gene’s expression (CLE18 and ICE2). Both of these genes are involved in different responses to environmental conditions, whose selective control could be highly beneficial to helping crops survive the ever harsher conditions caused by climate change. CLE18 is involved in root growth, and when overexpressed shows phenotypically longer and irregular roots. ICE2 helps to modulate several cold response genes and whose overexpression leads to increased cold tolerance in plants. If these genes are able to be selectively controlled, crops may have an increased chance of survival of snap freezes and extended droughts. gRNA’s A through E were created for each target gene as well as a multiplex guide that were all transformed into separate plants.
Demonstration of Engineering Success
Once transformed into A. thaliana, we initially screened for positive transformants by plating seeds onto Murashige and Skoog plates containing hygromycin as a selective factor. Hygromycin is an antibiotic that slows root growth of wild type plants. Positive transformants will be clearly visible on a plate of seeds due to its greener leaves and longer roots in comparison to their untransformed counterparts.
Once we identified positive transformants from our CLE18 guide B CRISPRa plates (n=2), they were carefully removed from the agar to be replanted in soil. Before replanting, an expected phenotype of increased root branching was observed. These changes were visibly different in comparison to the roots of wild type plants of the same age. The transformants were left to grow bigger to be able to conduct RT-qPCR analysis. Unfortunately, at the time of RNA extraction, RNA yield was not great enough to perform the RT-qPCR.
