Contribution by construction of an AlcR + CRISPRa plasmid (BBa_K4058011)

In consideration of the many issues posed by climate change, the 2021 Guelph iGEM team has decided to tackle the issue of increasing crop die off caused by the many drastic changes in local weather patterns emerging across the globe. Our contribution to the iGEM registry was centred around the idea to increase a plant's natural response to environmental stress. Our focus was turned to two specific contributors: drought and cold stress.

Using Arabidopsis thaliana as a model organism, our design involved the use of CRISPRa to target the following genes: CLE18, who’s over expression results in increased root length and ICE2, who’s over expression results in increased cold tolerance. Using specific gRNA’s to target these genes in complement with our AlcR + CRISPRa construct would in theory allow farmers to upregulate the expression of these genes in response to local weather predictions in preparation of expected cold snaps and droughts.

Figure 1: Scaffold of our CRISPRa scaffold with our AlcR ethanol inducible promoter. Important features include the gRNA scaffold with Bsa1 cut sequences, VP64 domain and hygromycin resistance region.

Our plasmid construct was successfully sequenced to confirm its identity. This plasmid was then successfully transformed into both E. coli and A. tumefaciens cells.

While this system was used by our team to target these specific genes, our AlcR + CRISPRa plasmid can be used by future iGEM teams in conjunction with their own unique gRNA scaffolds for the targeting of different genes specific to their own projects.

University of Guelph iGEM 2019