Riannon Smith; Melena Garrett, Utah State University
CRISPR (Clusters of Regularly Interspaced Short Palindromic Repeats) loci and cas (CRISPR associated) genes provide adaptive immunity against invasive elements such as viruses and plasmids in bacteria and archaea. Although the structure and function of CRISPR system Types I, II, III, V, and VI have been determined, the Type IV CRISPR systems have not yet been examined experimentally. To better understand the structure and function of Type IV CRISPR systems, we are constructing a plasmid to recombinantly co-express several Type IV CRISPR system proteins from Acidothiobacillus ferrooxidans. We hypothesize that recombinant co-expression in E. coli of all the Type IV genes (csf1, csf2, csf3, cas6) along with a CRISPR will allow for a multi-subunit RNA-guided surveillance complex to form in vivo. Using ligation-independent cloning Type IV genes csf1, csf2, csf3, and cas6 were inserted into transfer vectors with no tag, a Histidine-tag, and a Maltose Binding Protein tag. Traditional cloning methods were used to insert the csf1 gene from transfer vectors into a destination vector. Traditional cloning methods will be further used to move sequences from the transfer vector into destination vectors to construct polycistronic vectors containing all four genes.