by Gulnihal Tomur
Many people regard biology as simply trying to solve the mysteries of nature through observation. A newly emerging field called synthetic biology bridges the gap between engineering and biosciences through reconstructing already existing biological systems or fabricating entirely new ones by using artificial DNA synthesis technology. As synthetic biologists construct and insert manufactured genes in cells, they have two main goals in mind: either turn the cells into drug or biofuel factories or gain a better understanding of the roles different biological components play in naturally existing biological system. Synthetic biology allows scientists to reconstruct existing biological systems on a molecular level.
When asked to describe synthetic biology, Dr. Caleb Bashor of the BioScience Research Collaborative at Rice alludes to a famous quote from physicist Richard Feynman: “What I cannot create, I do not understand.” Dr. Bashor and his team attempt to rebuild intricate biological pathways to better comprehend their underlying principles. He refers to cells as micro-level machines — and “just like any machine, the cells have wiring inside them in the form of DNA and proteins.” Accordingly, Dr. Bashor’s approaches cellular systems from an engineering standpoint as he reconstructs these systems just like how an electrical engineer would rewire a machine to figure out how it works. “I am partly an engineer and partly a scientist, trying to understand how cells are organized so I can rewire them,” Dr. Bashor explained.
Yet, Dr. Bashor did not start his scientific journey as a synthetic biologist; instead, he first occupied himself with biochemistry as an undergrad at Reed College. He was especially drawn to thinking about biology from the protein structure scale. At UCSF, he pursued the study of biological structures as a grad student from a perspective rooted in biophysics. Dr. Bashor got involved in synthetic biology at its early stages through a protein structure and function lab at UCSF, where they put different protein domains together to change the function of the protein as it was expressed in the cell. Dr. Bashor shares how they were essentially engaged in synthetic biology, yet did not know how broad the scope of their research was: “Eventually, people came along and told us ‘You guys are doing synthetic biology.’ And our reaction was ‘What’s that?’” As Dr. Bashor immersed himself more and more into this field, he dedicated his postdoc research at MIT to engineering transcriptional networks in eukaryotic cells. His current research at BioScience Research Collaborative encapsulates an even more ambitious approach as he and his team establish “synthetic regulatory circuits to reprogram the behavior of human cells.” Dr. Bashor and his team currently tackle mammalian regulatory circuits with potential applications that range from biosensing to cell-based therapy.
In order to fully understand Dr. Bashor’s work and its implications, we must first look into a natural gene circuit. Both eukaryotes and prokaryotes carry multiple genes in their genome, some of which get turned on and off depending on the external and internal signals cells receive. Since most proteins have complex structures, usually multiple genes are clustered together in a transcription unit. This then codes for the subunits of a single protein. The expression of these genes are all dependent on a single promoter for their expression; the promoter serves as the site where RNA polymerase binds to DNA to initiate the transcription of the information embedded in DNA to mRNA. The so-called on-off switch of such transcription units is a specific DNA segment called an operator. Located either within or after the promoter, the operator is responsible for regulating when RNA polymerase can bind to DNA and start the transcription of the gene cluster. The promoter, the operator, and the clustered genes make up genetic circuits known as operons.
When another protein called a repressor, which is the product of a regulatory gene separate from the operon’s gene cluster, is present in its functional state, it binds to the operator, thus physically blocking RNA polymerase from binding to the promoter. Internal and external factors, such as the presence of a specific molecule, influence the activity of the repressor by changing its 3D shape, which allows cells to switch on and off specific gene clusters based on environmental cues. Synthetic biologists take advantage of this knowledge as they introduce molecules in the cell which may turn on or off a repressor, add artificially designed circuitry into the existing systems, or isolate certain parts of a naturally occurring gene circuit through genetic modification.
Mirroring and reconstructing such systems, however, still proves to be a real challenge despite the advancement in synthetic biology. One common problem scientists deal with is the instability of artificial gene circuits. These artificial circuits can break the natural balance of the metabolic systems of the cell. As the artificial gene circuits use the energy which would otherwise be used for growth and division, they hinder the cells from maximizing their fitness. Thus, any mutant cell in the cluster which did not accept the circuit outcompete the synthetically engineered cells. “The circuit becomes unstable evolutionary; the cells spit out the circuit over time in order to grow faster,” explains Dr. Bashor. Intrigued by this evolutionary process, he put his mind to engineering a circuit that doesn’t get spat out. He realized that he needed a device that would help him and his team monitor the evolutionary process of cells with artificial circuits in them. The result was eVOLVER — it enabled them to work with many circuits at a time. As an automated multi-culture platform, eVOLVER can control multiple parameters at once. It shows the cell growth and detects any disparities which may signal a mutant culture that spat out the circuit put into the system. “We can grow [the cells], watch the circuits break, and see where they recover their fitness as a result of the circuit breaking,” tells us Dr. Bashor. Ultimately, the device operates as a tool for being able to build circuits and then test their stability, which is a crucial factor if the circuit is desired to be stable for a long time so that, for instance, it can be used for the production of a chemical.
Another challenge scientists face presents itself in the complexity of multi-gene systems. Pleitropic genes, which influence two or more seemingly unrelated phenotypic traits, and gene clusters regulated by common factors hinder scientists who aim to understand the function of individual parts in such systems. Therefore, synthetic biologists turn to a technique called refactoring. “Refactoring is a term borrowed from computer science,” tells us Dr. Bashor, “somebody gives you a code that is really hard to work with, so you rewrite in a way that it is easier to change the parts individually and to see what you’re changing.” In a similar fashion, synthetic biologists “physically abstract genes away from their native genomic content.” (1) The new content in which scientists place the gene in question tends to be a simple plasmid. Not only does this new medium offer easier control over the effects and outcomes of the gene due to its simplicity, but it also allows scientists to quickly modify the new genomic content since putting a new piece of DNA in a plasmid is a relatively easy process. By eliminating the interference caused by the natural genomic context, synthetic biologists observe the role of specific genes in the whole system. Yet, Dr. Bashor warns us not to see interference as a sheer burden on the system. “You may make the regulation very simple, but then you lose something in the behavior. That’s a clue that the regulatory forces that are associated with that context are important.” In the end, he sees refactoring, and synthetic biology in general, as a trial-and-error process — synthetic biologists simplify and modify and rewire the systems they are curious about over and over again to master the role of individual parts, which then allow them to see the bigger picture more clearly.
There are many other obstacles in the synthetic biology scene scientists are still trying to overcome today. The inability to insert big chunks of DNA into cells, for example, is hindering scientists from taking full advantage of synthetic biology in cell therapy. Yet, Dr. Bashor sees the intersection of synthetic biology and cell therapy as one of the most promising developments to look out for in the future. As the trial-and-error process that is synthetic biology continues to ignite curiosity in current and future scientists, the field will offer more and more to the scientific community. From advancements in agriculture to biofuel production, the manipulation of biological systems has already enhanced our lives in ways we may not even realize, and it continues to help us finesse biological systems in ways we desire.
Sources
Synthetic Biology Explained. https://archive.bio.org/articles/synthetic-biology-explained (accessed Jan 20, 2020).
The Bashor Lab at Rice. http://bashorlab.rice.edu/index_large.html (accessed Jan 20, 2020).