Current use of microbes for metabolic engineering suffers from loss of metabolic output due to natural selection. Rather than combat the evolution of bacterial populations, we chose to embrace what makes biological engineering unique among engineering fields – evolving materials. We harnessed bacteria to compute solutions to the biological problem of metabolic pathway optimization. Our approach is called Programmed Evolution to capture two concepts. First, a population of cells is programmed with DNA code to enable it to compute solutions to a chosen optimization problem. As analog computers, bacteria process known and unknown inputs and direct the output of their biochemical hardware. Second, the system employs the evolution of bacteria toward an optimal metabolic solution by imposing fitness defined by metabolic output. The current study is a proof-of-concept for Programmed Evolution applied to the optimization of a metabolic pathway for the conversion of caffeine to theophylline in E. coli. Introduced genotype variations included strength of the promoter and ribosome binding site, plasmid copy number, and chaperone proteins. We constructed 24 strains using all combinations of the genetic variables. We used a theophylline riboswitch and a tetracycline resistance gene to link theophylline production to fitness. After subjecting the mixed population to selection, we measured a change in the distribution of genotypes in the population and an increased conversion of caffeine to theophylline among the most fit strains, demonstrating Programmed Evolution. Programmed Evolution inverts the standard paradigm in metabolic engineering by harnessing evolution instead of fighting it. Our modular system enables researchers to program bacteria and use evolution to determine the combination of genetic control elements that optimizes catabolic or anabolic output and to maintain it in a population of cells. Programmed Evolution could be used for applications in energy, pharmaceuticals, chemical commodities, biomining, and bioremediation.

/pdf/programmed-evolution-for-optimization-of-orthogonal-4710gvexjh.pdf

Programmed evolution for optimization of orthogonal metabolic output in bacteria.

The Vision and Change report recommended genuine research experiences for undergraduate biology students. Authentic research improves science education, increases the number of scientifically literate citizens, and encourages students to pursue research. Synthetic biology is well suited for undergraduate research and is a growing area of science. We developed a laboratory module called pClone that empowers students to use advances in molecular cloning methods to discover new promoters for use by synthetic biologists. Our educational goals are consistent with Vision and Change and emphasize core concepts and competencies. pClone is a family of three plasmids that students use to clone a new transcriptional promoter or mutate a canonical promoter and measure promoter activity in Escherichia coli. We also developed the Registry of Functional Promoters, an open-access database of student promoter research results. Using pre- and posttests, we measured significant learning gains among students using pClone in introductory biology and genetics classes. Student posttest scores were significantly better than scores of students who did not use pClone. pClone is an easy and affordable mechanism for large-enrollment labs to meet the high standards of Vision and Change.

pClone: Synthetic Biology Tool Makes Promoter Research Accessible to Beginning Biology Students.

Students often memorize the definition of a transcriptional promoter but fail to fully understand the critical role promoters play in gene expression. This laboratory lesson allows students to conduct original research by identifying and characterizing promoters found in prokaryotes. Students start with primary literature, design and clone a short promoter, and test how well their promoter works. This laboratory lesson is an easy way for faculty with limited time and budgets to give their students access to real research in the context of traditional teaching labs that meet once a week for under three hours. The pClone Red Introductory Biology lesson uses synthetic biology methods and makes cloning so simple that we have 100% success rates with first year students. Students use a database to archive their promoter sequences and the performance of the promoter under standard conditions. The database permits synthetic biology researchers around the world to find a promoter that suits their needs and compare relative levels of transcription. The core methodology in this lesson is identical to the core methodology in the companion Introductory Biology Lesson by Campbell and Eckdahl. The methods are reproduced in both lessons for the benefit of readers. The two CourseSource lessons provide the detailed information needed to reproduce the pedagogical research results published in CBE Life Sciences Education by Campbell et al., 2014.

Using Synthetic Biology and pClone Red for Authentic Research on Promoter Function: Genetics (analyzing mutant promoters)

Synthetic biology is a new field that combines engineering principles, mathematical modeling, and molecular biology techniques to design and construct novel biological parts, de- vices, and systems with applications in energy, medicine, environmental science, and technol- ogy We discuss examples of mathematical models aiding in biological investigations, biology aiding in mathematical investigations, and the two fields working together in synthetic biology to attack some of the world's biggest problems Opportunities abound for mathematicians to contribute to this budding field 1 INTRODUCTION In 1975, 140 scientists and ethicists convened a summit at Asilomar to discuss guidelines for research in the cutting-edge field of genetic engi- neering Recombinant DNA technology had suddenly made it possible to fundamen- tally and permanently modify organisms by inserting functional elements from other organisms Today, the methods of recombinant DNA technology are the foundation for another emerging field with the potential to revolutionize science on a similar scale Synthetic biology is a diverse and rapidly growing field, only about a decade old, and all practitioners do not necessarily agree on a definition for the field For our pur- poses, synthetic biology is the application of engineering principles and mathematical modeling to the design and construction of biological parts, devices, and systems with applications in medicine, environmental science, technology, and energy Synthetic biology is distinguished from the related foundational field of genetics by its broad, systems-level approach, and from systems biology by its focus on building new systems rather than studying existing ones Synthetic biologists often think like engineers, using the principles of abstraction and modularity to guide the design of new parts, devices, and systems Modular designs are supported by standardization of assembly methods, so that DNA parts built according to the standards are inter- changeable Descriptions of these parts are listed in an online catalog (16), and the parts themselves are freely available Mathematical tools from a spectrum of subfields play an important part in synthetic biology research As expected in mathematical biology, mathematical modeling and analysis can help scientists understand biological processes Perhaps surprisingly, in synthetic biology, the converse is also true: Biological models and representations can help scientists solve mathematical problems In this article we discuss some examples of each direction of this interaction between mathematics and biology Finally, we highlight a number of recent synthetic biology projects in which many disciplines, including mathematics and computer science, have come together to attack some of the world's biggest problems 2 MATHEMATICAL MODELING IN THE DESIGN OF SYNTHETIC BIOLOGICAL SYSTEMS Our knowledge about biological systems has increased dramatically in the so-called genomic era, when it became possible to sequence entire organisms But knowing the sequence of A's, C's, G's, and T's that make up a bac- terium, or a mouse, or a human, is just one step in understanding how genes function

https://www.jstor.org/stable/10.4169/amer.math.monthly.121.09.857

Synthetic Biology: A New Frontier

Unnatural amino acid (UAA) incorporation is an invaluable technique that is seeing increased use. THG73 is an amber suppressor tRNA used to incorporate > 100 residues at the UAG, amber stop codon, in Xenopus oocytes. We have found that yeast Phe frameshift suppressors (YFFS) can incorporate UAAs at the CGGG and GGGU quadruplet codons in vitro and in vivo, allowing simultaneous incorporation of three UAAs in the nicotinic acetylcholine receptor (nAChR). The YFFS are more “orthogonal” than the amber suppressor tRNA, THG73, but the frameshift suppressors incorporate UAAs less efficiently than THG73. A library of tRNAs derived from THG73 has produced an amber suppressor that is “orthogonal” and suppresses similarly to THG73. An analogous opal suppressor tRNA allows incorporation of UAAs at the UGA, opal stop codon. The use of the amber, opal, CGGG, and GGGU codons should allow for the simultaneous incorporation of four UAAs in vivo. Bioorthogonal labeling of UAAs is useful for the addition of large fluorophores. We incorporated p-AcPhe at α70 of the nAChR and labeled with biotin and Cy5.5 hydrazide. Biotin and Cy5.5 hydrazide consistently labeled three proteins on oocytes not expressing α70p-AcPhe and isn’t useful for site-specific labeling of ketone containing UAAs in oocytes. We explored the known subunit stoichiometry of the nAChR (2α:β:γ:δ) expressed in oocytes and detected each subunit with the HA tag by Western blot. The α-subunit is present in excess of the other subunits in a ratio of ≈ 3:1, which is expected to be 2:1. UAAs are being sold commercially for detection of protein-protein interactions in eukaryotic cells. The UAAs are heterogeneously incorporated and little is known about the effect on protein function and stability. We heterogeneously incorporated UAAs into the nAChR and detected changes in function by shifts in EC50. Many UAAs altered the function of the nAChR. Incorporation of photo-reactive UAAs allowed for detection of cross-linking by Western blot. Heterogeneously incorporated UAAs also altered the functional nAChR expression on the surface of oocytes. Site-specific and heterogeneous incorporation of multiple UAAs are useful techniques for novel experiments to explore protein function, FRET experiments, cross-linking, and protein expression.

In Vivo Incorporation of Multiple Unnatural Amino Acids

Introduction: We investigated frameshift suppressor tRNAs previously reported to use five-base anticodon-codon interactions in order to provide a collection of frameshift suppressor tRNAs to the synthetic biology community and to develop modular frameshift suppressor logic devices for use in synthetic biology applications. Results and Discussion: We adapted eleven previously described frameshift suppressor tRNAs to the BioBrick cloning format, and built three genetic logic circuits to detect frameshift suppression. The three circuits employed three different mechanisms: direct frameshift suppression of reporter gene mutations, frameshift suppression leading to positive feedback via quorum sensing, and enzymatic amplification of frameshift suppression signals. In the course of testing frameshift suppressor logic, we uncovered unexpected behavior in the frameshift suppressor tRNAs. The results led us to posit a four-base binding hypothesis for the frameshift suppressor tRNA interactions with mRNA as an alternative to the published five-base binding model. Conclusion and Prospects: The published five-base anticodon/codon rule explained only 17 of the 58 frameshift suppression experiments we conducted. Our deduced four-base binding rule successfully explained 56 out of our 58 frameshift suppression results. In the process of applying biological knowledge about frameshift suppressor tRNAs to the engineering application of frameshift suppressor logic, we discovered new biological knowledge. This knowledge leads to a redesign of the original engineering application and encourages new ones. Our study reinforces the concept that synthetic biology is often a winding path from science to engineering and back again; scientific investigations spark engineering applications, the implementation of which suggests new scientific investigations.

Bacterial Logic Devices Reveal Unexpected Behavior of Frameshift Suppressor tRNAs

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Bacterial Logic Devices Reveal Unexpected Behavior of Frameshift Suppressor tRNAs