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The Whole Scheme
In order to be able to hack the genetic programming of a biological system, we need two main tools: i) a DNA delivery system and ii) a DNA suppression system. Both, the delivery and the supression systems should be very specific so that other organisms can not be damaged.
For the DNA delivery system we chose a P1-based phagemid [1] which is only able to recognize E. coli. The lytic state of this system only works in the presence of arabinose, making possible for us to “fabricate” a solution containing the bacteriophage to be used to hack cells in situ. Given that the lytic state remains in the off state upon arabinose induction, hacked cells are able to survive in any environment.
The DNA suppression system we chose makes use of TALENs and ZFNs [2]. Both, TALENs and ZFNs, are engineered proteins able to recognize specific sequences and bind to them. A nuclease attached to the binding domain then is capable to cause a nick in the DNA. If coupled strategically, these proteins can be used to digest the DNA as restriction enzymes.
After we joined the delivery and the suppression systems, the whole scheme ended as shown in Figure 1. Plasmids containing TALEN/ZFN sites must be used to program organisms intended to be released into the environment. If any organisms shall be hacked, the first step would be to generate a bacteriophage solution in the laboratory. This solution will contain the bacteriophages at the required concentration, and the bacteriophages themselves will contain a phagemid with the appropriate TALEN/ZFN gene and the new program, if any.
In our case, fluorescent proteins were selected to represent the programs and monitor the hacking process (Figure 1B). Variable reporters can be used to monitor the hacking process in situ.
References
[1] Kittleson JT, DeLoache W, Cheng HY, Anderson JC. (2012) Scalable Plasmid Transfer using Engineered P1-based Phagemids. ACS Synth Biol. 1:583-9.
[2] Strauβ A, Lahaye T. (2013) Zinc fingers, TAL effectors, or Cas9-based DNA binding proteins: what's best for targeting desired genome loci? Mol Plant. 6:1384-7.
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