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From 2011.igem.org

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Overall project

This year's goal of iGEM Warsaw team is to develop 2 foundational techniques for synthetic biologists:

Our first goal is to make easy and efficient protocol for cell free cloning. It skips most basic step of classical cloning approach - plasmid propagation in bacteria or yeast. This allows cloning of constructs that are toxic to host cells (i.e. nucleases or lysins) and speeds up the whole procedure at least three times. The cell free cloning process involves rolling circle amplification of ligation products by phi29 polymerase. Our protocol is speed-oriented so we'll try to optimize all time consuming steps: DNA digestion, substrate purification, ligation and product amplification. We'll also try to deal with specificity problems of phiX29 polymerase by designing RNA primers optimized for BioBrick amplification.

Our second goal is to provide BioBrick community standardized method of insertion of large constructs into E. coli genome via landingPad mediated homologous recombination. To achieve this we are going to develop BioBrick-compatible landingPad vectors and at least one recombination proficient host strain with LPs already in genome. We are also going to utilize the power of flippase to achieve very tight expression regulation and allow induced genome editing (deletions/substitutions).