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Problem Statement

The plasmid currently available in the market for gene insertion at a specific target sequence has been successfully used in tagging proteins in mammalian cells but costs a few thousand dollars. The design will attempt to generate plasmids through the use of Bacterial Artificial Chromosomes (BACs) that are antibody-specific for tagging fission yeasts, and are available for a few hundred dollars. These newly designed plasmids must be cost-efficient and versatile enough that a researcher can easily manipulate them as desired, allowing substitutions for alternative selectable markers to express multiple strains of yeast.

Project Design

The entire genome of fission yeast (S. pombe) is available in the form of a Bacterial Artificial Chromosome (BAC) library. Once obtained, the epitope tagging (TAG) sequence will be inserted at the desired locus. The TAG sequence will need to be accompanied by a selectable marker to identify the correctly tagged proteins in E. coli. Another marker should also be expressed in fission yeast (S. pombe) so that modified S. Pombe organisms can be identified for this marker before being studied using the epitope tag. The figure below shows a schematic of this design:

Bacterial Artificial Chromosomes

Alternative Designs

Use of RNAi- The larger underlying problem with genetic engineering is that it is not amenable to S. pombe or many other eukaryotic organisms because of its reliance on homologous recombination. The Non-Homologous End-Joining (NHEJ) Pathway, which is controlled by several genes including NEJ1, is known to suppress homologous recombination in both S. pombe and mammalian cells. If a method was developed to suppress the NHEJ pathway, homologous recombination could be greatly enhanced in any species. Once this was done, gene manipulation could be done using a site-specific plasmid, the same way it is done for budding yeast S. cerevisiae or for embryonic stem cells.

Subcloning- DNA subcloning is the technique used for the insertion of foreign DNA into a vector through the use of restriction enzymes and DNA ligase. The technique is one of the fundamental methods in molecular biology, and it is used widely. An alternative design would be to use restriction enzymes and DNA ligase to insert the gene sequence of choice into a plasmid that can be cloned and is compatible with many organisms, such as S. pombe and E. coli.