Optimized protocols and plasmids for in vivo cloning in yeast

Area of Science:

• Molecular Biology
• Yeast Genetics
• Synthetic Biology

Background:

• Saccharomyces cerevisiae is a powerful tool for in vivo DNA cloning and plasmid construction.
• Traditional gap repair and in vivo cloning methods, while efficient, can necessitate extensive screening of resulting clones.
• Pre-selection strategies using Escherichia coli-based blue/white screening have been previously shown to improve efficiency.

Purpose of the Study:

• To demonstrate the utility of blue/white selection in Saccharomyces cerevisiae for pre-selecting positive clones during plasmid assembly.
• To show that this strategy can be applied to assemble plasmids from multiple ectopic DNA fragments simultaneously.
• To enhance the versatility of shuttle plasmids by extending their multi-cloning sites.

Main Methods:

• Utilizing Saccharomyces cerevisiae for gap repair and in vivo cloning.
• Employing shuttle plasmids with blue/white selection capabilities in Escherichia coli for pre-selection.
• Introducing multiple ectopic DNA fragments into yeast cells via a single transformation step.
• Extending the multi-cloning sites of existing shuttle plasmids.

Main Results:

• The blue/white selection strategy effectively allows for the pre-selection of positive clones in yeast.
• This method successfully facilitates the assembly of plasmids from several ectopic DNA fragments in a single transformation.
• The multi-cloning sites of three shuttle plasmids were expanded to include fifteen additional restriction enzyme recognition sites.

Conclusions:

• The integration of blue/white selection into yeast-based cloning significantly streamlines the identification of desired plasmids.
• This enhanced yeast cloning system simplifies the assembly of complex plasmids from multiple DNA fragments.
• The expanded multi-cloning sites provide greater flexibility for downstream subcloning and vector manipulation.