What is this “spies and bloggers” metaphor you keep using?

Synthetic microbes can either report back on their own behavior or their own perceptions (bloggers), or they can report on their environmental conditions or the behavior of microbes around them (spies).  Not all environmental microbes are genetically tractable enough to make into bloggers, and in some cases the microbes whose behaviors we are interested in grow so slowly that they are not appropriate for laboratory experiments.  In these cases we use model genetically tractable organisms as spies instead (for example E. coli, Bacillus, Roseobacter, or Shewanella species).  Spy organisms can then monitor the flux of biologically important molecules through their environment.

What exactly do you mean when you say that you are “DNA bar coding” organisms?

Synthetic biologists use the term “DNA bar code” as a metaphor for inserting a  bit of unique, extra DNA into an organism in response to a particular event (e.g. experiencing horizontal gene transfer, constructing a biofilm, or detecting an environmental chemical like a nutrient or a communication molecule).  This creates a record in an organism's DNA, making it possible to later determine through sequencing what fraction of a population has experienced this event. Usually this extra DNA is nonfunctional, but in some cases it can cause the microbe to release a signal (e.g. producing green fluorescent protein). 

Synthetic biologists use the metaphor of “bits” when they talk about engineering memory into organisms.  What does this mean, exactly?

Synthetic biology can be used to trigger microbes to rewrite portions of their DNA, for example using enzymes called integrases.  These enzymes reverse segments of DNA in response to a trigger, creating a physical, hardwired memory in an organism’s experiences.  This DNA flipping is analogous to the flipping of bits in a computer’s memory between 0’s and 1’s.  When synthetic biologists talk about “bits of genetic memory” they are talking about the number of individual DNA sequences that can be independently flipped in one organism’s DNA to create a record.  Each DNA flip is analogous to flipping a computer’s memory from a 0 to a 1, and creates a new memory state within an organism.

Why do you use methyl halides?  Why not use something even more inert, like the traditional oceanographic tracers SF6, or CFCs?

There is no existing genetic code for the production of the traditional, highly inert oceanographic tracers like SF6 and CFCs, which means that we cannot simply swap code for the production of these gases into genomes.  Because no specific gas reporter system is perfect, we are in the process of developing a palette of other gases to broaden the utility of biosensors in the environment. 

I’ve read that methyl bromide is a fumigant used in strawberry agriculture.  Won’t it harm the organisms in your samples?

CH3Br is used in high concentrations in agriculture to kill pathogenic fungi in soil.  Our organisms produce CH3Br in concentrations that are 3 orders of magnitude lower than those needed for fumigation.  While there are no reports of any effects of CH3Br on bacteria, we still test for this in each organism we modify. For every new construct we test to make sure that the host organism does not experience any negative metabolic effects from the introduction of new DNA or from the presence of Br- or CH3Br in its environment.

Doesn’t adding this gas expression gene into the host organism affect its ability to grow? 

In theory it is possible that adding new DNA into an organism could create a metabolic burden, slowing its growth.  We test for this by comparing the growth rates of wild-type and engineered organisms to insure no negative effects from either the introduction of new DNA or from changes in environmental conditions.

Won’t these organisms be dangerous if released into the environment?

None of our experiments involve the release of any organisms into the environment.  The fate of all our biosensors is destruction in our autoclaves.  In addition, we scrupulously comply with all university, local, state, and national regulations surrounding DNA modification.

What if your biosensors get eaten by other microbes in your samples? 

For some of our experiments this is not an issue because we do not need mixed populations to explore the effects of abiotic conditions on microbial sensing, behavior, and reproduction.  For example, we can study the effects of nutrient variation on the ability of microbes to trade plasmids, or we can study the ability of microbes to detect nutrients as a function of physical or chemical conditions in their environment.  In these experiments there are no other microbes, so there is no risk of biosensors being eaten or outcompeted by more robust wild organisms.

When we put biosensors in living communities in the laboratory we can correct for any changes in population size via using the ratiometric gas approach (see acronyms below).  In this approach biosensors make 2 gases: one which reports constantly (a constitutive reporter), and a second which reports conditionally (a logical reporter).  The ratio of these two gives a signal independent of cell population. 

There seems to be a lot of lingo in the synthetic biology field. 

Acronyms:

HGT: horizontal gene transfer

AHL: acyl homoserine lactones, a class of signaling molecules used ubiquitously by Gram negative bacteria to quorum sense.

MGE: mobile genetic elements

MHT: methyl halide transferase, an enzyme that produces methyl halides using the methyl group on S-adenosylmethionine and a halide ion from the cytosol.

Definitions:

Mobile genetic elements: A general category that comprises all genetic material within a cell that can move, including DNA-transposable elements (transposons), plasmids, chromids (Petersen 2013 citation) and bacteriophage” (Nature link) see Petersen 2013

Conjugative plasmid: a plasmid that can be transferred through direct cell-cell transfer.

Transconjugant: cells that have received a conjugative plasmid

Integrase memory: Integrase memory is one type of DNA memory system, relying on the inducible expression of an integrase enzyme.  Once expressed, the integrase targets specific DNA recognition sequences and inverts the DNA sequence between those recognition sites.

Integrase: An integrase is an enzyme that inverts DNA between specific sites.

Ratiometric reporter: A signaling system with two outputs.  One indicates cell number (ie is constitutively expressed) and the other represents expression of an inducible promoter.  This class of reporters allows normalization of biosensor signal to biosensor cell population.

Inducible switch: this is a promoter that can be induced to express a downstream reporter.

Constitutive expression: A constitutive signal is expressed continuously, and can be used as an indication of viable cell number.