Demonstration of Engineering Success

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.

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.

Wildtype 3: Wildtype Arabidopsis thaliana growing on MS plates.
CLE18 gB 3: MS + hygromycin plate screening for positive transformants of our CLE18 guide B CRISPRa seeds. Hygromycin in the plates slows the growth of any seeds that did not successfully uptake the plasmid. Positive transformants can be visually seen on the plate due to their greener leaves and longer roots.

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.

Wildtype 1: Wildtype Arabidopsis thaliana after growth on the MS plates. Root systems show no branching/irregularity and similar or shorter root length in comparison to our CLE18 guide B CRISPRa transformants.

CLE18 gB 2: Visible phenotypic change in the root systems of our positive CLE18 guide B CRISPRa transformants. Over expression of this gene leads to increased root length and root irregularity in comparison to the wildtype A. thaliana root system. Roots of the stronger plant appear to be approximately 1cm without being fully extended with increased branching from the main lateral root.

University of Guelph iGEM 2019