Pichia

High levels of cell growth, foreign protein production, organized golgi bodies, inexpensiveness, ability to be inducible, and post-translational modifications allow P. pastoris to be an ideal vector of choice when compared with others such as Saccharomyces. The ability to isolate proteins because of its low endogenous protein secretion also contributes to our interest in making the vector. P. pastoris also has a high growth rate with two inducible promoters allowing methanol as its key source of carbon and energy. Although many successful experiments have used E.coli as their expression system, P. pastoris has become increasingly popular. P. pastoris serves as a better host when attempting to express foreign genes because the yeast can perform post-transational modifications, folds proteins properly, tightly regulate transcription via its wide range of promoters, and be purified easily. The Pichia system is an ideal construct for producing vaccines. We have designed a standard Pichia system that can be used for the production of various different vaccines. In theory, a high yield circuit can be established that only requires the exchange of the variable antigen, while leaving the remaining factors constant. In the created vector we have designed a promter pGAP to be altered to IGEM standards by the MSC linker piece and plan to ligate in RFP and eventually express the desired protein. 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. In the above figure the gel picture indicates a successful ligation of the multiple cloning site (MCS). A cocktail restriction digest was done in order in confirm correct ligation. The ligation of the RFP into pGAP was done. Linearization was supposed to be the next step however we were not able to get the enzymes needed on time to do the linearization. The vector will then be transformed into Pichia competent cells. 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 that ability to be inducible.

Agro

Integration of the modified Ti plasmid into plants begins with first creation of a binary system containing a gene of interest could potentially be advantageous to both plants and animals. These modified plasmids will remove the ability of Agrobacterium to produce tumors and introduce genes that could potentially give plants the capability to live and prosper in environments that these plants typically could not live in. Our goal was to re-engineer the Ti Plasmid by introducing genes that code for red fluorescent protein (RFP) and a advantageous gene of interest. DNA containing pORE Expression Series Vector (20I) and RFP was hydrolyzed and extracted from the 2012 iGEM kit plate. Electrocompetent Escherichia coli cells were first used to manipulate the plasmid due to the relative ease to grow and cultivate E. coli. The RFP gene was successfully inserted into the plasmid of E. coli using electroporation (Figure 3). The transformed E.coli cells were allowed to incubate for 24 hours @ 37ºC (Figure 4). After incubation, plasmid extraction was done using QIAprep Miniprep to remove the plasmid and quantify the purity of their DNA. Restriction digestion using the restriction enzymes EcoR1 and Pst to visually analyze how big the RFP fragment sizes are. Due to the time it takes for the plants to grow, verification of the function of our plasmid remains untested. Promptly after sufficient amount of growth occurs, our A. tumefaciens carrying our modified plasmid will trans-infect the Brassica Rapa plants. A positive result will be seen if RFP is expressed in the plants. Further testing will also be done to check how well the plants re-uptake the Ti plasmid. We recently started working with the pPZP500 cloning vector but as of now we do not have any data collected on this vector. The vector was provided to us by Dr. Holger Bohlmann, and Ali Muhammad Amjad from the University of Natural Resources and Applied Life Sciences in Vienna.

Works sited Weidner,, Maria, Marcus Taupp, and Steven J. Hallam “Expression of recombinant proteins in Methylotrpohic Yeast Pichia Pastoris.” PubMed.gov.Pubmed, 25 Feb.2010. Web 25. 2010 Coutu, C., Brandle, J., Brown, D., Brown, K., Miki, B., Simmonds, J., & Hegedus, D. D. (2007). pORE: binary vector. Transgenic Research, 16(6), 771–781. doi:10.1007/ s11248-007-9066-2 Cubero, J., Martínez, M. C., Llop, P., & López, M. M. (1999). A simple and efficient PCR method for the detection of Agrobacterium tumefaciens in plant tumours. Journal of Applied Microbiology, 86(4), 591–602. doi: 10.1046/j.1365-2672.1999.00700.x Gelvin, S. B. (2003). Agrobacterium-Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool. Microbiology and Molecular Biology Reviews, 67(1), 16–37. doi:10.1128/MMBR.67.1.16-37.2003