Pichia

This year, we accomplished getting pgapz alpha digested to retrieve the part we needed. The vector was gel isolated through gel excision. Following the isolation, the vector was ligated as well as the MCS linker. We then transformed the host into ecoli and colonies were picked. Mini preps were analyzed and a digest was done of pGap. Purification and linearization was done with EcoRI and Pst. We were then able to ligate RFP and pGapz alpha. The vector will then be transformed into Pichia. Future possibilities would include expressing a protein that can be useful as well as advancing uses of Pichia over Ecoli because of its ability to perform post translational modifications which is important in eukaryots. Also, we would like to work on the pPik 9 because of it being inducible.

Agro

Agrobacterium tumefaciens is a gram-negative soil bacterium that is the causative agent of Crown-Gall disease in both dicotyledon and monocotyledon plants. The tumors caused by Crown-Gall disease contain segments from the Tumor Inducing (Ti) Plasmid known as the TDNA, which completely integrates with the host's genome. In genetic re-engineering, a binary vector system is used because the Ti plasmid is over 250 kb and is too large to easily manipulate. Binary vectors consist of small plasmids with a cloning site and a selectable maker gene between the left and right border of the TDNA making it easier to manipulate. Modification of a gene of interest and insertion into the Ti Plasmid of A. tumefaciens allows exploitation of the natural ability to A. tumefaciens to transmit DNA. Creation of a binary system containing a gene of interest could potentially be advantageous to both plants and animals.