How to Grow Almost Anything

Week 11: Genome Engineering

Making a small contribution to the Media Lab's CRISPR mission


Class protocol reference

PAM-SCANR: identifying the PAM sequence of novel Cas enzymes

The PAM-SCANR system involves 3 plasmids, constructed through kinase-ligase-dpnI (KLD) assembly, that are sequentially transformed into E. coli, each with its own antibiotic resistance to enable selection for E. coli that have all 3 plasmids. These plasmids express: 1) the Cas enzyme to be characterized 2) the gRNA, complementary to the target sequence 3) from 5' to 3' - the target sequence followed by a randomized 8-mer (NNNNNNNN) for the Cas enzyme to bind to, followed by the LacI promoter, LacI, and GFP cassette (repressed by LacI). When the Cas binds to the 8-mer, it physically blocks transcription of LacI, allowing GFP to be expressed. The GFP-expressing bacteria can then be sorted and selected via flow cytometry (FACS) and sequenced to identify the PAM sequence. I'm still waiting on the results of my Cas's sequencing.

Assignment Questions

    • What tasks must a cell perform to live? A cell must be able to make and use ATP; produce the necessary enzymes via transcription, translation, and post-translation modifications; transport enzymes and small molecules to their destination; destroy infectious agents.
      What bacterial innate immune mechanisms must be overcome to perform genome transplantation? Bacterial CRISPR-Cas systems and restriction enzymes have evolved to cut foreign DNA.
      Can you suggest an alternative to genome transplantation to achieve genome scale engineering of bacterial genomes? Methylation patterns, and thus gene expression, in bacteria can be controlled through temperature of growth conditions.
      What mammalian innate immune mechanisms must be overcome to efficiently install large DNA molecules in mammalian cells? Exocytosis and RNAi.