Open Sequence Initiative: a part submission standard to complement modern DNA assembly techniques

The discipline of synthetic biology emphasizes the application of engineering principles such as standardization, abstraction, modularity, and rational design to complex biological systems. The archetypical example of such standardization is BioBrick RFC[10], introduced in 2003 by Tom Knight at MIT. BioBricks are stored on a standard plasmid, pSB1C3, which contains prefix and suffix sequences flanking the DNA sequence specifying a biological part. The prefix and suffix sequences contain two pairs of 6 base-pair (bp) restriction enzyme sites (EcoRI+XbaI and SpeI+PstI), which can be used for both part assembly and quality control. BioBricks are intended to be well- characterized biological parts, such as genes or promoters, that function in a predictable fashion and can be readily combined to make complex systems. The rules of the RFC[10] BioBrick assembly method require that none of the restriction sites used in the prefix and suffix be present in the parts themselves. This requirement can be an onerous imposition for iGEM teams developing large, novel parts, such as genes or entire operons that are obtained by amplifying DNA sequences from environmental samples or microorganisms. While iGEM teams may use methods such as site-directed mutagenesis to remove illegal restriction sites from a part's sequence, it is certainly possible that this mutation will alter the functionality of the part – a very undesirable outcome. In addition, the mutagenesis of illegal restriction sites is an unnecessary burden on teams, given the limited time and resources available to teams during each year’s iGEM competition. Efforts spent mutagenizing sites would be better spent characterizing and improving parts. This RFC proposes an alternative submission standard to eliminate these problems

Tetraspanin CD53 promotes lymphocyte recirculation by stablising L-selectin surface expression

Tetraspanins regulate key processes in immune cells; however, the function of the leukocyterestricted tetraspanin, CD53 has remained unknown. Here we show that CD53 is essential for lymphocyte recirculation. Lymph nodes of Cd53-/- mice were smaller than wild-type mice due to a marked reduction in B cells and a 50% decrease in T cells. This reduced cellularity reflected an inability of Cd53-/- B and T cells to efficiently home to lymph nodes, due to the near absence of L-selectin from Cd53-/- B cells and reduced stability of L-selectin on Cd53-/- T cells. Further analyses, including on human lymphocytes, showed that CD53 inhibits L-selectin shedding via both ADAM17-dependent and -independent mechanisms. The disruption in lymphocyte recirculation in Cd53-/- mice led to impaired immune responses dependent on antigen delivery to lymph nodes. Together these findings demonstrate a previously unrecognized essential role for CD53 in lymphocyte trafficking and immune responses

The impact of synthetically expanded genetic codes on evolvability

Synthetic biology allows researchers to address previously unanswerable biological questions through the wholesale reengineering of living systems. Even one of the most fundamental properties of biology – the canonical genetic code for the translation of genetic information into proteins – may now be altered to include nonstandard amino acids (nsAAs) because synthetic incorporation systems for nsAAs now match native host machinery in terms of efficiency and fidelity. However, the evolutionary stability of these systems and the potential for them to promote the evolvability of the organisms in which they are deployed remains mostly unexplored. In chapter one of this dissertation, I explore how Escherichia coli strains that incorporate a 21st nsAA at the recoded amber (TAG) stop codon evolve resistance to the antibiotic rifampicin. I found a variety of mutations that lead to substitutions of nsAAs in the essential RpoB protein confer robust rifampicin resistance, and I interpret these results in a framework in which an expanded code can increase evolvability in two distinct ways. I consider the implications of these results for the evolution of alternative genetic codes, in particular their support for the codon capture model of genetic code evolution. In chapters 2 and 3, I employ bacteriophage T7, which utilizes the translation apparatus of its E. coli host, as a model organism for whole-genome evolution with an expanded genetic code. I show that phages evolved on an E. coli host that incorporates 3-iodotyrosine became dependent on an alternative genetic code as they adapted to higher fitness. In particular, phages with a 3-iodotyrosine substitution in the type II holin protein outcompete phages with canonical amino acids at this position. Inspired by this surprising outcome, I performed massively parallel evolution experiments in T7 with six expanded genetic codes, finding over a thousand substitutions to nsAAs in these experiments, many of which displayed genomic signatures of positive selection. Two of the nsAAs accumulated in genomes at a rate comparable to a mock genetic code expansion with the canonical amino acid tyrosine. Together these results show that expanded genetic codes alter functional sequence space and create new possibilities for adaptive mutations, thereby improving evolvability in these organisms.Cellular and Molecular Biolog

Decaffeination and Measurement of Caffeine Content by Addicted <i>Escherichia coli</i> with a Refactored <i>N</i>‑Demethylation Operon from <i>Pseudomonas putida</i> CBB5

The widespread use of caffeine (1,3,7-trimethylxanthine) and other methylxanthines in beverages and pharmaceuticals has led to significant environmental pollution. We have developed a portable caffeine degradation operon by refactoring the alkylxanthine degradation (Alx) gene cluster from <i>Pseudomonas putida</i> CBB5 to function in <i>Escherichia coli</i>. In the process, we discovered that adding a glutathione <i>S</i>-transferase from <i>Janthinobacterium</i> sp. Marseille was necessary to achieve <i>N</i>₇-demethylation activity. <i>E. coli</i> cells with the synthetic operon degrade caffeine to the guanine precursor, xanthine. Cells deficient in <i>de novo</i> guanine biosynthesis that contain the refactored operon are ″addicted″ to caffeine: their growth density is limited by the availability of caffeine or other xanthines. We show that the addicted strain can be used as a biosensor to measure the caffeine content of common beverages. The synthetic <i>N</i>-demethylation operon could be useful for reclaiming nutrient-rich byproducts of coffee bean processing and for the cost-effective bioproduction of methylxanthine drugs

Control of oxidative phosphorylation by vitamin A illuminates a fundamental role in mitochondrial energy homoeostasis

The physiology of two metabolites of vitamin A is understood in substantial detail: retinaldehyde functions as the universal chromophore in the vertebrate and invertebrate eye; retinoic acid regulates a set of vertebrate transcription factors, the retinoic acid receptor superfamily. The third member of this retinoid triumvirate is retinol. While functioning as the precursor of retinaldehyde and retinoic acid, a growing body of evidence suggests a far more fundamental role for retinol in signal transduction. Here we show that retinol is essential for the metabolic fitness of mitochondria. When cells were deprived of retinol, respiration and ATP synthesis defaulted to basal levels. They recovered to significantly higher energy output as soon as retinol was restored to physiological concentration, without the need for metabolic conversion to other retinoids. Retinol emerged as an essential cofactor of protein kinase Cδ (PKCδ), without which this enzyme failed to be activated in mitochondria. Furthermore, retinol needed to physically bind PKCδ, because mutation of the retinol binding site rendered PKCδ unresponsive to Rol, while retaining responsiveness to phorbol ester. The PKCδ/retinol complex signaled the pyruvate dehydrogenase complex for enhanced flux of pyruvate into the Krebs cycle. The baseline response was reduced in vitamin A-deficient lecithin:retinol acyl transferase-knockout mice, but this was corrected within 3 h by intraperitoneal injection of vitamin A; this suggests that vitamin A is physiologically important. These results illuminate a hitherto unsuspected role of vitamin A in mitochondrial bioenergetics of mammals, acting as a nutritional sensor. As such, retinol is of fundamental importance for energy homeostasis. The data provide a mechanistic explanation to the nearly 100-yr-old question of why vitamin A deficiency causes so many pathologies that are independent of retinoic acid action.—Acin-Perez, T., Hoyos, B., Zhao, F., Vinogradov, V., Fischman, D. A., Harris, R. A., Leitges, M., Wongsiriroj, N., Blaner, W. S., Manfredi, G., Hammerling, U. Control of oxidative phosphorylation by vitamin A illuminates a fundamental role in mitochondrial energy homoeostasis