On Plasmids

Genome synthesis for the rest of us

Who synthesizes whom?
© Dr Gopal Murti / Photo Researchers, Inc.

Ever since I’ve been a practicing molecular biologist, we’ve used plasmids as vehicles for genetic engineering. Or, more accurately, we’ve used the entire range of extragenomic information that can replicate inside cells. That would include viral vectors, which have been harnessed for use in both prokaryotic and eukaryotic cells. I sometimes wonder whether this predilection to use episomes has driven perceptions of how synthetic biology should be carried out. If you want to engineer a cell it would seem easiest to add the extragenomic information in a somewhat orthogonal fashion, with its own origin and its own means of being maintained, distinct from the chromosome. I think this is one of the reasons that the “Venter shunt,” synthesizing the whole genome, has attracted so much attention (other than the obvious,...

That said, I think that we are beginning to see a much more realistic idea coming into vogue: the genome as the unit of synthetic engineering. What can the hoi polloi do? Well, in the distant past, the way was pointed by folks who originally did directed evolution of cells. Barry Hall, now director of the Bellingham Research Institute in Bellingham, Washington, and emeritus professor at the University of Rochester in New York, is a pioneer here, with a clever directed-evolution study that led to a new beta-galactosidase (ebg) evolved from an otherwise seemingly unused gene in the E. coli. Willem “Pim” Stemmer of Maxygen, is under-recognized (from my point of view) for his early successes in genome shuffling, also known as DNA breeding, which is a form of directed evolution that allows functional variation to be recombined at fantastic rates, well in excess of nature. This includes the über-scary shuffling of HIV genomes. (Amusingly, the synthesis of poliovirus drove folks nuts, while Stemmer’s earlier work on creating new viruses didn’t raise much of an eyebrow in the popular press.)

I think that we are beginning to see a much more realistic idea coming into vogue: the genome as the unit of synthetic engineering.

More recently, George Church at the Harvard Medical School has developed multiplex-automated genomic engineering (MAGE), which allows large-scale, iterative alteration of the E. coli genome using synthetic oligonucleotides. Folks have also recognized that naturally competent organisms such as Acinetobacter may provide traction for genome engineering.

Soon there will be tailored genomes that contain augmented genetic codes. And MAGE or its relatives will begin to be used regularly for phenotype evolution. For example, could you evolve the entire genome of a bacterium so that it was completely dedicated to overproducing a given, industrially important protein? I lived through the use of directed evolution for nucleic acids and proteins, and now we’ll have an age of directed evolution of cellular genomes. The corresponding readouts available from NextGen sequencing will make the analysis of the products of directed evolution relatively straightforward. And the tools currently used by systems biologists will allow us to make sense of the glut of information that will result. It could actually be argued that directed evolution is perhaps the best means of more fully understanding the interconnections that are being discovered by systems biologists.

It could actually be argued that directed evolution is perhaps the best means of more fully understanding the interconnections that are being discovered by systems biologists.

I’m now wondering whether in the age of directed evolution we’ll answer this question: what does the fitness landscape for a cell look like? Now, that’s a loaded question, given that fitness landscapes will be highly dependent upon the environment in which the cell finds itself. A cell (or protein or nucleic acid) in a laboratory environment has a very different available landscape than a cell (or protein or nucleic acid) in the wild. Still, it’s a legitimate question. One would almost have to believe that cells live on very large neutral plains. The genomes of cells have had to learn to get along over billions of years. If you’re the odd gene out, you get booted pretty quickly. That level of interconnection is probably only possible if the landscape for one-mutation moves is very broad.

Of course, there are many places where one can climb or one can fall on the landscape, but my guess is that the new peaks (or nadirs) are relatively mild. I think that the directed evolution of cells will not produce the same range of phenotypes as the directed evolution of proteins. This would seem paradoxical, given that cellular machinery is for the most part made of proteins. Let me refine that musing: phenotypically, you will get more bang for your mutational buck by focusing on a single protein rather than the cell as a whole. You will eat lactose better if you mutate beta-galactosidase, rather than mutating a genome.

And this nicely brings me back to the start. On the one hand I think that genomes should be the unit of evolution, since they are already an integrated system. On the other, I think that the integrated system will only grudgingly provide new function. And hence, we need to provide that new function orthogonally, either by having facile integration methods for new information (cells have already figured this out of course; look to see how pathogenicity islands work) or via…plasmids.

This column is adapted from “Andy Ellington’s Blog” which can be accessed at ellingtonlab.org/blog

Andrew Ellington is the Fraser Professor of Biochemistry at the University of Texas at Austin. He works on way too many things, including the directed evolution of functional nucleic acids, molecular computation schemes, and engineering translation. He is not a synthetic biologist, but he plays one at conferences. He has a decided interest in biodefense, which generally stems from his not wanting to have his eyeballs bleed out.

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