rahma zakaria
how to grow almost anything
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genome engineering


  1. What tasks must a cell perform to live?
      I think that to call a cell "alive," it must be able to replicate when supplied with sufficient nutrients and placed in appropriate conditions. For example, if you have colonies of cells on an agar plate, and you try to culture the cells in an overnight culture, I think the colonies would be considered living cells if they can grow in the culture (provided with the nutrients in LB and incubated at 37C), and they'd be considered dead if they could no longer grow in the culture. Also, bacteriophages are not considered to be living because they cannot replicate their own DNA.
  2. What bacterial innate immune mechanisms must be overcome to perform genome transplantation?
      As discussed in class: CRISPR-based targeting, restriction enzymes, nucleases, interferon response, exocytosis, and RNAi.
  3. Can you suggest an alternative to genome transplantation to achieve genome scale engineering of bacterial genomes?
      Perhaps causing the cell to produce a faulty polymerase and seeing if any living cells are produced.
  4. What mammalian innate immune mechanisms must be overcome to efficiently install large DNA molecules in mammalian cells?
      Codon bias of bacterial vs. mammalian cells, and antigen tagging/other aspects of the innate immune system.


  5. identifying pam sites for engineered cas-9 enzymes


    We used PAM-SCANR to select for cells expressing GFP due to binding of their PAM sequence to a library of random sequences, then identified the PAM by sending the cells for sequencing.

    Electroporating cells with the plasmid encoding the engineered Cas9 enzymes and an antibiotic resistance marker.