Voigt paper

So the basic idea here was to show that they could set up a genetic circuit using the invasin gene as an 'output module' -- that is, the piece of the circuit that actually affects the environment in some way. Invasin is an E. coli protein that binds to proteins called beta-1-integrins on the surface of a number of mammalian cell types and can induce bacterial uptake by the mammalian cells. So the authors thought, well, wouldn't it be nice if you could, for example, set up a genetic circuit that caused invasin to bind selectively to tumor cells?

This was pretty painstaking, and boring to read about, but the end result was interesting. So they're obviously careful about genetic characterization of invasin, first of all. They checked whether invasin required other E. coli adhesion systems in order to bind to cell surfaces (it didn't), then showed that inv+ E. coli were able to successfully invade a few different human cancer cell lines (they examined osteosarcoma, hepatocarcinoma, and HeLa cells). The bacterial recovery rates were different for the different cancer types, possibly because of varying expression of beta-1-integrins on the different cell types.

Next they constructed a arabinose-inducible circuit for invasin. So first what they discovered is that background transcription of this circuit's promoter was actually sufficient for invasin synthesis, and to reduce this background expression, they altered the ribosome binding site of the circuit: they replaced the 5' untranslated region with randomized sequence and also randomized the first base of the first and second codons. So to figure out what they got from the resulting library, they did a positive selection for the inducible phenotype, then a negative screen to check which of this subset kept on invading without induction.

So next they linked the invasive phenotype with an anaerobic environment. Their strategy was the same as before: alter the ribosome binding site, generate a library, then do a positive selection and negative screen. Next they note that, since a cancerous environment tends to attract a whole bunch of different bacterial species to it, it would be nice if they could link the invasive phenotype with bacterial cell density. Specifically, quorum sensing, a method by which bacterial cells can communicate with each other. What they did was find an existing genetic switch (called lux), and created a plasmid where the inv gene was placed under the control of the lux promoter. They found pretty much what you'd expect -- when inv was expressed via a constitutive promoter, invasion was independent of cell density, where under the lux-promoter's control, it was detectable only at high bacterial cell densities.

So, this is actually a pretty cool idea. Follow-up work, and I think a good line for comments, would be along the lines of, well ok, so invasin gets you into tumor cells, but now what do you do? Also you could use this sort of genetic circuit to as a vector for a number of different applications aside from targeting cancer cells.