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Dec 1, 2013 (Vol. 33, No. 21)

The Rise of Systems Biology

  • Co-opting Biological Systems

    “We developed the ability to synthesize almost any molecule that we would like to, but one great challenge for the field is transitioning from individual molecules to networks that function together inside the living cells,” explained Virginia W. Cornish, Ph.D., professor of chemistry.

    As part of early efforts to co-opt directed evolution to build new molecules inside cells, Dr. Cornish and colleagues linked the chemistry of enzyme catalysis to cell survival. “We envisioned that we could do this by including small molecule chemistry into the yeast two-hybrid assay. This involves selecting for enzymes that catalyze the synthesis or the cleavage of a bond based on the transcription of an essential reporter gene and coupling two small molecules,” Dr. Cornish said.

    This concept, which initially took advantage of the high-affinity interaction between methotrexate and dihydrofolate reductase, provided the possibility of exploiting orthogonal chemistry in a robust way inside living cells, and is currently being widely used for many applications, such as assays for controlled protein degradation.

    The proof of principle for linking enzyme catalysis to reporter genes exploited cephalosporin hydrolysis by the Enterobacter cloacae P99 cephalosporinase, which was incorporated into a three-hybrid system to measure cephalosporinase activity in vivo as a change in lacZ transcription, and this screen also helped isolate wild-type cephalosporinase from a pool of inactive mutants.

    The promises offered by yeast genetics emerged as an attractive experimental possibility. “As we started taking advantage of the budding yeast as an organism in which to study directed evolution, we sought ways to perform mutagenesis in this setting,” said Dr. Cornish. While Dr. Cornish and colleagues initially exploited homologous recombination as a mutagenesis strategy, they saw the dependence on transformation emerge as a shortcoming.

    “We addressed this by placing the DNA on a heritable cassette plasmid, embedding it in endonuclease sites, and performing endonuclease cleavage to create double-stranded breaks and recruit the homologous recombination machinery, and this helped generate reasonably large libraries,” Dr. Cornish explained.

    Synthetic and systems biology promise to profoundly impact the field of biosynthetic engineering. “Increasingly, rather than make drugs by chemical synthesis, we can engineer yeast and other organisms to biosynthesize intermediates on which we can do further chemistry,” he continued.

    In a current project, Dr. Cornish and colleagues are using engineered yeast as a low-tech biosensor for cholera. Receptors that recognize cholera were engineered on the surface of yeast, and the yeast were engineered to turn on a plant pigment. “We can, in this way, envision a very cheap and safe product that we can hand out into local communities for the detection of cholera by nontechnical people in the community,” Dr. Cornish added.

  • Single-Cell Analysis

    “When we take a complex tissue and perform expression profiling, we do not necessarily know the cellular origin of the molecules that were quantified,” says assistant professor Peter A. Sims, Ph.D. In cellular populations, biological processes are unlikely to occur in a synchronous fashion, and this opens significant challenges for analyzing those processes and interpreting the data.

    One approach to address this shortcoming is to perform analyses at the single-cell level. “We wanted to look at the transcriptomes of individual cells,” explained Dr. Sims. Examining many genes across large numbers of individual cells provides an ideal way to catch a glimpse of biological processes at the single-cell level, and concomitantly capture interindividual heterogeneity in the population.

    Ideally, single-cell analysis should be performed by direct detection, to avoid the consequences of amplification bias, and the approach should be time-efficient and affordable. “At this time, we do not have a technology that does not compromise the number of cells analyzed in favor of the number of targets analyzed and vice versa, so it is very difficult to look at a lot of genes and a lot of cells with one tool,” said Dr. Sims.

    Investigators in Dr. Sims’ lab rely on two approaches for single-cell analysis, microfluidics and microscopy. Microfluidics uses very small reagent volumes and consequently reduces contamination, whereas microscopy provides additional visual information about the system being examined at a specific time.

    By using soft lithography microfluidics, a technology that allows single-cell behavior to be captured under a broad number of conditions, Dr. Sims and colleagues have developed tools for single-cell transcriptome analysis. One of these tools, targeted probe-based expression profiling, offers the possibility of simultaneously exploring several tens of genes within the same cell. “The advantage of this approach is that we are able to probe the transcriptome at the location where the cell is observed,” noted Dr. Sims.

    With each cell in its own picoliter-sized chamber, the array can be sealed with a glass surface that is chemically functionalized to allow the capturing of the RNA, which can be reverse transcribed. “We are also developing another tool that uses microarray platforms for large-scale, genome-wide RNA sequencing, where we can generate thousands of cDNA libraries on the microarray well chip,” Dr. Sims said.

    Systems biology has made it possible to test hypotheses that merely a few years ago were beyond the reach of experimental approaches. Nonetheless, by working at the juncture of biomedicine, biotechnology, and the clinic, scientists are extending their reach, unveiling mechanistic details, and hastening paradigm shifts. Central to these endeavors, and one of the fundamental teachings that has emerged thus far, is the power of science’s integrative nature.

  • To the Microbiome...and Beyond!

    Click Image To Enlarge +
    A team led by Boston University’s Dr. James Collins collected numerous E. coli expression profiles and then used systems biology to develop whole-scale genome models to gain novel insights on how antibiotics work. [fusebulb/Fotolia]

    “We are focusing on bacterial and human systems, and we are beginning to study the interface of the two, the microbiome, from a synthetic biology perspective,” says James J. Collins, Ph.D., professor of biomedical engineering at Boston University. Dr. Collins served as keynote speaker at Columbia’s recent symposium.

    Many advances in systems and synthetic biology have become reality thanks to landmark developments, and one of these was the construction of transcriptional switches that co-opted RNA regulatory functions. A significant stride came in 2004, when Dr. Collins and colleagues reported the development of a post-transcriptional Escherichia coli RNA-based regulator that was able to either suppress or activate gene expression.

    In this model, a cis-repressive sequence inserted upstream of a ribosome binding site formed a stem-and-loop structure at the 5'-untranslated region of the mRNA, interfering with gene expression. Also, a small noncoding RNA molecule expressed in trans was able to target the repressed RNA, activating gene expression. Subsequently, Dr. Collins and colleagues developed genetic counters. With this approach, several RNA switches are serially activated by output from the previous switch, allowing user-defined induction transcriptional output events to be counted.

    These advances opened the need to generate synthetic circuits that can be programmed to also kill a cell that has been rewired with a new function. By using two lytic proteins, Dr. Collins’ lab developed a programmable kill switch. “After three public hearings, the Presidential Commission for the Study of Bioethical Issues highlighted the kill switch as a much-needed safeguard,” said Dr. Collins.

    In addition to safety, this system offered other advantages. “After we published this work, several biotech companies showed interest in using this technology for combating corporate espionage,” explained Dr. Collins. They feared that competitors could obtain engineered microbial strains. This concern is alleviated if a microorganism is endowed with the property to self-destruct when programmed to do so.

    “What really has been driving my lab is going after antibiotics,” remarked Dr. Collins. The need to develop new antimicrobial agents, or to devise new approaches to address antimicrobial resistance, is fueled by the increasing number of resistant strains, a trend that is compounded by the decreasing number of newly developed and approved antimicrobial agents.

    “We collected hundreds of E. coli expression profiles, and used systems biology to develop whole-scale genome models to gain new insights into how antibiotics work,” said Dr. Collins. Experiments on quinolones revealed that the DNA damage response network was a key participant in bacterial response. The oxidative damage response network was also induced, however, and studies on additional antibiotics revealed that they can also alter cellular metabolism and affect cellular respiration as part of their mechanisms to cause bacterial cell death.

    “We tried to use these findings to boost the killing efficacy of antibiotics,” added Dr. Collins. In a high-throughput assay that examined over 2,000 compounds, Dr. Collins and colleagues showed that the bactericidal activity of antibiotics was improved with RecA inhibitors. Subsequently, the use of bacteriophages overexpressing LexA3, a noncleavable version of the SOS system repressor LexA, boosted from 100- to 10,000-fold the in vitro killing efficacy of bactericidal antibiotics, and illustrated the possibility of using engineered bacteriophages as antibacterial adjuvants.

    Moreover, in the first animal study in systems biology, the antibiotic-enhanced bacteriophage in combination with quinolone treatment rescued 80% of the mice from death. “Much of what is happening in synthetic biology is still at the microbial stage, and we would like to move this field toward higher organisms,” concluded Dr. Collins.

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