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Ethan's Research: Yeast Genome Integration and Bacterial Secretion of Kumamolisin

Along with Guy, I am researching methods of yeast cloning via integration into the yeast genome. For those of you who don't know, yeast undergoes a process called recombination. Recombination likens to crossing over in that homologous DNA sequences on the plasmid insert and portions of the yeast genome swap strands. Unlike crossing over, however, yeast genomic integration does not occur exclusively during meiosis. This makes yeast perhaps the best chassis for genomic integration, since it provides a naturally-occurring and highly efficient integration system. Like crossing over, efficiency of gene retention decreases as the homologous sites move further away from the centromere.

Yeast System

• This PDF provides a quick summary of the methods available for transformation into yeast.
• This is a more detailed version.
• A System of Shuttle Vectors and Yeast Host Strains Designed for Efficient Manipulation of DNA in Saccharomyces ceratisiae

Yeast Integrative Plasmid Vectors (YIp)

• GAL1 and GAL10 (galactose catabolism) selection
• TRP1 selection

Prokaryotic System

Promoters

• BBa-J23100 (strong strength)
• BBa-J23108 (medium strength)
• BBa-J23109 (weak strength)

Terminators

• BBa-B0015

RBS

• Constitutive

Secretion

• HlyA Gene Sequence
• A novel C-terminal signal sequence targets Escherichia coli haemolysin directly to the medium
• Analysis of the haemolysin transport process through the secretion from Escherichia coli of PCM, CAT or beta-galactosidase fused to the Hly C-terminal signal domain
• Analysis of the haemolysin secretion system by PhoA-HlyA fusion proteins

Kumamolisin Info

• 57 kDa (beta-galactosidase: 540 kDa; source)
• 1701 bp
• 567 aa