Team:Vanderbilt/Project/Background
Abstract
Every system that is genetically engineered harbors a potentially fatal vulnerability. The source of life's great diversity - spontaneous mutation - is for the synthetic biologist the source of constant apprehension and risk. The relentlessness of genetic mutation has discouraged previous attempts at ever overcoming this perpetually looming menace. Whether in a multifaceted genetic circuit or a simple protein expression platform, mutation is inevitable, and once it disrupts function, the organism will no longer experience the burden of transgene expression, causing the mutant to outcompete whatever has the intended sequence. Evolution and mutation work hand in hand to select against the maintenance of synthetic DNA sequences. Indeed, the mantra has been that time in the form of mutation and evolution will always find a way to erode and ultimately destroy everything that an engineer builds, no matter how ingeniously it may designed.
This year, here at Vanderbilt iGEM, we are fighting back. We are proposing a novel approach based on rationally designed genomic architectures that promises to offer synthetic biologists unprecedented control over the evolutionary stability of their creations. At the heart of our strategy is an advanced computational algorithm that integrates decades worth of scientific data identify and correct the highly-mutation prone 'hotspots' that lurk in every gene. Our strategy has a strong foundation in a rich literature from the fields of cancer biology and others that have annotated and characterized mutation hotspots for almost every conceivable source of mutagen, from ultraviolet radiation to recombination to polymerase errors.
When combined with synthetic DNA technology, our process becomes a simple and reliable optimization that is universally applicable to any coding gene being expressed in any organism. Our project first demonstrates the power of this rational synthetic gene design strategy by employing several canonical as well as highly original protocols for assaying DNA damage and its effects on the stability of artificial genetic elements. From these techniques, we can quantify everything from the selective subset of mutation types occurring on an in vitro level, up to how mutational loss of function translates at the scale of populations of genetically modified organisms.
To complement our work, we have harnessed our algorithm for use in what is becoming one of the most important tools for engineering biological molecules: directed evolution experiments. Not only can our engineered changes increase evolutionary stability in applications such as transgenic bioreactors, but it can also construct gene sequences that are more prone to mutate, thus accelerating studies into how to use evolutionary selection to produce tailored functional modifications to proteins.
Finally, we have investigated ways to build new genetically modified organisms that exhibit greatly increased resistance to mutation. Combining our sequence-based strategies with the introduction of exogenous genes and removal of endogenous genes has enabled us to produce an expression platform for synthetic genes that not only has enhanced DNA repair mechanisms, but also has an entire artificial pathway introduced for the elimination of mutant strains from a population.
While any single engineered change to reduce mutation may still fail, when our innovative approaches to modulating evolutionary stability are taken in combination, they offer an unprecedented hope for taming evolutionary entropy. More than a victory for synthetic biology, we prove that through rational design principles- exactly what mutation most virulently tries to uproot- and with enough clever innovations, it is possible to defend against what seemed like an inevitability of nature. Score one for engineering.
Importance of Genetic Stability
The implications of reducing evolutionary potential are far-reaching. All of synthetic biology is impacted by the instability of genes, but there are several areas where it is of particular concern, namely manufacturing, medicine, and biosafety.
In terms of production, bioreactors are especially vulnerable to gene mutation due to the metabolic stress exhibited by the cells in such an environment. The constant push for maximum production gives an evolutionary advantage to any cells that spontaneously mutated to produce less or no product. An entire incubator could be overtaken by these mutated cells leading to a loss in money, time, and resources. In a medical context where pharmaceutical agents are produced, a mutation can have even worse consequences of creating a deleterious drug that would be harmful, such as antibody-producing strain of cells that undergoes a spontaneous mutation leading to unintended interactions. Furthermore, on a cellular level, changes in a genome leading to differing phenotypes is a problem encountered in breweries. Brewers often keep frozen stocks of particular cell lines in order to not keep using the same population which can mutate over the course of many brewing cycles.
The advent of gene therapy which allows insertion of genes directly into living cells poses parallel difficulties in terms of ensuring continued function. The cell will not only try to get rid of the foreign DNA because of its associated metabolic load, the sequence will be prone to mutations that either cause a nonfunctional or dysfunctional protein product.
Similarly, genetically modified organisms that are introduced to the environment have the possibility to escape their intended area as well as transfer their genes to other organisms. To combat this issue, many have developed sophisticated killswitch circuits, but what has not been addressed is the degradation of this safety mechanism which is subject to more evolutionary pressure than other components due to its lethality. Both the circuit and the genes composing it are subject to mutation resulting in inactivation and the spread of GMO. (cite Haynes)
All of these issues are almost entirely due to the effect of metabolic load and selective pressures on genes which cause them to either be inactivated or deleted. It has been demonstrated that increasing the metabolic load of the cell decreases its rate of replication which is directly correlated to its Darwinian fitness. Cells that have to produce are less adapted to their environment and are more likely to be outcompeted in a heterogeneous population where other cells are not under the same load. (cite Ellis) Moreover, the load of translating active proteins that create their own secondary metabolites compounds the stress on the cell leading to further decreased fitness.
The metabolic load itself can eliminate the cells ability to survive, but there is an even greater danger posed by increasing cellular load which is stress-induced mutagenesis. The stress of augmented rate of protein production strains the cell into initiating a global response system that changes gene expression and thereby cell metabolism. This includes pathways that increase mutation potential by up-regulating error prone DNA polymerases and down-regulating error correcting enzymes as well as encouraging the movement of insertion sequences. This mutator state under stressful conditions from increased metabolic load is important for adaptive evolution but is counterproductive to the goals of synthetic biologists attempting for stable expression. (cite Foster)
Strategies to Combat Mutation
We took a three-pronged approach to minimize mutation by both repairing mutations that occur and preventing mutagenesis from happening. Our strategies target the three levels at which genetic constructs are implemented in engineered biological systems- the physical DNA sequence, its genomic and circuit context, and lastly the entire organism which is host to the synthetic DNA.
