From genetics to biotechnology: Synthetic biology as a flexible course-embedded research experience.

The need for changing how science is taught and the expansion of undergraduate research experiences is essential to foster critical thinking in the Natural Sciences. Most faculty research programs only involve a small number of upper-level undergraduate students each semester. The course-based undergraduate research experience (CURE) model enables more students to take ownership over an independent project and experience authentic research. Further, by creating projects that fit into a curriculum's learning goals and student-oriented outcomes, departments help strengthen critical thinking skills in the classroom. Here, we report on the incorporation of a synthetic biology CURE into a mid-level cellular biology course and two advanced level genetics/molecular biology courses. Synthetic biology involves systematic engineering of novel organisms, such as bacteria and plants, to work as functional devices to solve problems in medicine, agriculture, and manufacturing. The value of synthetic biology and its ultimate utility as a teaching tool relies on reusable, standard genetic parts that can be interchanged using common genetic engineering principles. This Synthetic biology CURE effectively achieves five essential goals: (1) a sense of project ownership; (2) self-efficacy: mastery of a manageable number of techniques; (3) increased tolerance for obstacles through challenging research; (4) increased communication skills; and (5) a sense of belonging in a larger scientific community. Based upon our student assessment data, we demonstrate that this course-based synthetic biology laboratory engages students directly in an authentic research experience and models important elements of collaboration, discovery, iteration, and critical thinking.

Engineered device in E. coli lyses S. aureus at physiological fever temperatures.

Multiple strains of Staphylococcus are resistant to antibiotics, including the well-known methicillin-resistant Staphylococcus aureus (MRSA). We share an engineered plasmid device in Escherichia coli that lyses the disease-causing pathogen, S. aureus. The device was engineered using BioBrick parts obtained from the International Genetically Engineered Machine foundation (iGEM). The cI-blue-lysostaphin device consists of a temperature-sensitive promoter that is activated under physiological fever temperatures above 35°C that drives expression of a blue chromoprotein reporter and mature truncated lysostaphin enzyme. The functioning cI-blue-lysostaphin device was tested for optimal lysis conditions in MM294 and DH5α E. coli chassis and across incubation temperatures ranging from 30-42°C. We conclude that the lysostaphin activity of the cI-blue-lysostaphin device differs between chassis and increases with greater incubation temperature.

Closed Genome Sequence of Yavru, a Novel Arthrobacter globiformis Phage.

We characterized the complete genome sequence of actinobacteriophage Yavru (Siphoviridae), a cluster FE bacteriophage infecting Arthrobacter globiformis NRRL B-2979; it was 89.5% identical to cluster FE phage Whytu, with a capsid width of 50 nm and a tail length of 90 nm. The genome was 15,193 bp in length, with 23 predicted protein-coding genes.

Merlin, the product of the Nf2 tumor suppressor gene, is an inhibitor of the p21-activated kinase, Pak1.

The Nf2 tumor suppressor gene codes for merlin, a protein whose function has been elusive. We describe a novel interaction between merlin and p21-activated kinase 1 (Pak1), which is dynamic and facilitated upon increased cellular confluence. Merlin inhibits the activation of Pak1, as the loss of merlin expression results in the inappropriate activation of Pak1 under conditions associated with low basal activity. Conversely, the overexpression of merlin in cells that display a high basal activity of Pak1 resulted in the inhibition of Pak1 activation. This inhibitory function of merlin is mediated through its binding to the Pak1 PBD and by inhibiting Pak1 recruitment to focal adhesions. This link provides a possible mechanism for the effect of loss of merlin expression in tumorigenesis.

Cellular transformation by a FERM domain mutant of the Nf2 tumor suppressor gene.

Mutations in the Nf2 tumor suppressor gene lead to tumor formation in humans and mice and cellular overproliferation phenotypes in Drosophila. The Nf2 encoded protein, merlin, shares close sequence similarity in its amino terminus to members of the band 4.1 family of membrane-cytoskeletal linkers. Similarities between merlin and this family suggest a role for merlin in regulating cytoskeletal function. However, the mechanism of the tumor suppressing activity of merlin is not yet understood. Mutational analysis of Nf2 in flies has led to the identification of a dominant-negative allele, which harbors mutations in the amino terminus of the protein. Here, we report that expression of a murine analog of this amino-terminal mutant of Nf2 leads to complete transformation of NIH3T3 fibroblasts in culture. Cells that express this Nf2 mutant allele display disruptions of the actin cytoskeleton, lack of contact inhibition of growth, and anchorage-independent growth. Finally, fibroblasts that express this mutant Nf2 allele form tumors when injected into nude mice.

Merlin phosphorylation by p21-activated kinase 2 and effects of phosphorylation on merlin localization.

The Nf2 tumor suppressor gene product merlin is related to the membrane-cytoskeleton linker proteins of the band 4.1 superfamily, including ezrin, radixin, and moesin (ERMs). Merlin is regulated by phosphorylation in a Rac/cdc42-dependent fashion. We report that the phosphorylation of merlin at serine 518 is induced by the p21-activated kinase PAK2. This is demonstrated by biochemical fractionation, use of active and dominant-negative mutants of PAK2, and immunodepletion. By using wild-type and mutated forms of merlin and phospho-directed antibodies, we show that phosphorylation of merlin at serine 518 leads to dramatic protein relocalization.