A. Malcolm Campbell

Evaluation of Three Automated Genome Annotations for Halorhabdus utahensis

Genome annotations are accumulating rapidly and depend heavily on automated annotation systems. Many genome centers offer annotation systems but no one has compared their output in a systematic way to determine accuracy and inherent errors. Errors in the annotations are routinely deposited in databases such as NCBI and used to validate subsequent annotation errors. We submitted the genome sequence of halophilic archaeon Halorhabdus utahensis to be analyzed by three genome annotation services. We have examined the output from each service in a variety of ways in order to compare the methodology and effectiveness of the annotations, as well as to explore the genes, pathways, and physiology of the previously unannotated genome. The annotation services differ considerably in gene calls, features, and ease of use. We had to manually identify the origin of replication and the species-specific consensus ribosome-binding site. Additionally, we conducted laboratory experiments to test H. utahensis growth and enzyme activity. Current annotation practices need to improve in order to more accurately reflect a genomes biological potential. We make specific recommendations that could improve the quality of microbial annotation projects.

Programmed Evolution for Optimization of Orthogonal Metabolic Output in Bacteria

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.

Figure Facts: Encouraging Undergraduates to Take a Data-Centered Approach to Reading Primary Literature.

Figure Facts is a versatile instructional tool designed to help college students tackle complex data figures in the primary literature.

Implementing Recommendations for Introductory Biology by Writing a New Textbook

A new introductory biology textbook responds to national calls for reform and promotes critical learning gains in scientific, quantitative, and metacognitive ability.

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

The synthetic biology lab module pClone is ideal for beginning students to conduct authentic research—inexpensive and easy to prep. Students clone and characterize promoters and share their results in research-grade databases with significant learning in the core concept of information and several core competencies described in Vision and Change.

Bacterial computing

Undergraduate students find that a genetically engineered machine can solve Hamiltonian Path Problems.

Improving the Lac System for Synthetic Biology

Abstract. The Escherichia coli lac Operon is controlled by a regulatory system that has been the subject of intensive study for the past fifty years. The system creates metabolic efficiency by responding to the levels of environmental lactose. In the absence of lactose, the LacI protein acts as a repressor of transcription from the lac promoter. Transcription begins when lactose binds to LacI, which results in the expression of three genes involved in lactose uptake and catabolism. The lac promoter is the most commonly used promoter in the field of synthetic biology. Although it is widely used, the lac promoter is known to have leaky transcription, meaning that transcription takes place even when the repressor is present and the inducer is absent. In an effort to redesign the lac promoter, we tested pLac variants that were reported to have a higher affinity for RNA polymerase than the wild-type. We also compared three mutants of the LacI repressor that were reported to have increasing affinity for the pLac promoter. Using GFP reporter constructs, we found that the pLacIQ1 promoter showed much higher levels of transcription than the wild-type promoter. Of the twelve combinations of promoters and repressors tested in the presence and absence of an inducer, we discovered that the wild-type LacI repressor protein with the pLacIQ1 mutant promoter is the best combination for high levels of induction and low levels of leaky transcription. Our results promise to help synthetic biologists design and build systems with tighter regulatory control.

Assembly of Standardized DNA Parts Using BioBrick Ends in E. coli

Synthetic biologists have adopted the engineering principle of standardization of parts and assembly in the construction of a variety of genetic circuits that program living cells to perform useful tasks. In this chapter, we describe the BioBrick standard as a widely used method. We present methods by which new BioBrick parts can be designed and produced, starting with existing clones, naturally occurring DNA, or de novo. We detail the procedures by which BioBrick parts can be assembled into construction intermediates and into biological devices. These protocols are based on our experience in conducting synthetic biology research with undergraduate students in the context of the iGEM competition.

IDENTIFICATION AND DNA SEQUENCE OF A NEW H+-ATPase IN THE UNICELLULAR GREEN ALGA CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)

Insertional mutagenesis was used to identify genes involved in mating and/or zygote formation in the unicellular green alga Chlamydomonas reinhardtii Dangeard. Approximately 800 insertionally mutagenized transformants were examined, and a single nonagglutinating mutant was identified. Plasmid rescue was used to clone a genomic fragment containing transforming DNA. This fragment was then used to identify the wild‐type copy of the gene disrupted during mutagenesis. The wild‐type gene is transcribed during all stages of the life cycle and, based on sequence similarity, encodes a P2‐type proton transporting ATPase. The gene is referred to as Pmh1 for plasma membrane H+‐ATPase. PMH1 displays the greatest sequence similarity to ATPases from two parasitic flagellates and a raphidophytic alga but not to the ATPase from a closely related green alga. We propose that PMH1 represents a distinct H+‐ATPase isoform expressed in flagellates.

Encouragement for Faculty to Implement Vision and Change

A two-semester introductory biology course that incorporates the recommendations of Vision and Change is described The course has been well received by students and produced good student learning outcomes. This essay demonstrates that Vision and Change’s recommendations are feasible and students welcome the improvements.