User:SKaret/Notebook/GroupProj/MoCloNotes1
Modular cloning strategy - Moclo
⁃ uses the Type IIS restriction enzymes BsaI and BpiI/BbsI to efficiently assemble up to six DNA fragments at a time.
⁃ this methods exploits the ability of Type IIS enzymes to cut outside their recognition site, and permits DNA fragments with compatible overhangs to be efficiently assembled.
⁃ engineer unique enzyme recognition sites that flank a DNA module in an inverse orientation, so that multiple DNA components can directionally assemble in a single reaction, while retaining only a defined 4bp fusion site in between
Steps:
1. insert fragments of DNA containing basic parts (promoters, UTRs, coding sequences, terminators, etc) into individual Level 0 plasmids, or choose from a growing number of libraries containing pre-constructed standardized modules.
2. compatible Level 0 vectors are directionally assembled into a Level 1 vector creating a single transcriptional unit (Ex: a promoter, 5’UTR, coding region, and terminator)
3. Joining up to six Level 1 modules —> Level 2 vector (functional genetic circuit). Flexibility has been built into the Level 2 vectors to allow for additional iterations of Level 1 assembly if necessary.
4. Combining multiple Level 2 vectors in the final assembly —> complex constructs constrained only by the ability of E. coli to maintain the final plasmid after transformation.
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MoClo Plasmid Kits Available from Addgene
1. EcoFlex MoClo Toolkit (deposited by Paul Freemont's lab)
⁃ The EcoFlex Toolkit is a collection of plasmids that features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins for use in E. coli for a variety of application
2. MoClo-YTK (deposited by John Dueber's lab)
⁃ A set of 96 standardized and characterized parts that can be used for bottom-up hierarchical assembly of single and multi-gene constructs for S. cerevisiae expression.
3. CIDAR MoClo Parts Kit (deposited by Douglas Densmore's lab)
- Collection of modular DNA parts and enhanced MoClo protocols to enable rapid one-pot, multipart assembly, combinatorial design and expression tuning in E. coli
