Team:UChicago/Experiments
In order to accomplish our goal in the laboratory, we first mutagenized an existing iGem backbone (psB1C3) using QuickChange PCR. This step introduces a new restriction cut site (HpaI) that is utilized later in the experiment, thus creating our new plasmid psB1C3mut. The plasmid has chloramphenicol resistance, so we grow it in this antibiotic to ensure that our plasmid of interest has been transformed into the bacteria/yeast.
The lab we work in provided us with a plasmid, pOW1, which contains SCARG4 (a gene that produces arginine) and PARS2 (an autonomous replicating element). We PCRd the region containing these two genes. Using Gibson assembly, we added these two genes into psB1C3mut, resulting in psD000. The presence of SCARG4 and PARS2 in our construct means that psD000 is a shuttle vector (it can be transformed and expressed into both E. coli and yeast).
We PCRd yEmRFP from a plasmid in the Glick lab (pOW1-yEmRFP) and ligated it into psD000. We plan to test whether this resulting plasmid (hopefully psD001) contains our genes of interest, and also to perform a series of fluorescence and sectoring assays which will show the expression of this vector in the yeast and its progeny. Once we know we have psD001, we can go about putting potential centromeric sequences into the plasmid.
The lab we work in provided us with a plasmid, pOW1, which contains SCARG4 (a gene that produces arginine) and PARS2 (an autonomous replicating element). We PCRd the region containing these two genes. Using Gibson assembly, we added these two genes into psB1C3mut, resulting in psD000. The presence of SCARG4 and PARS2 in our construct means that psD000 is a shuttle vector (it can be transformed and expressed into both E. coli and yeast).
We PCRd yEmRFP from a plasmid in the Glick lab (pOW1-yEmRFP) and ligated it into psD000. We plan to test whether this resulting plasmid (hopefully psD001) contains our genes of interest, and also to perform a series of fluorescence and sectoring assays which will show the expression of this vector in the yeast and its progeny. Once we know we have psD001, we can go about putting potential centromeric sequences into the plasmid.
