To Plate or to Simply Unfreeze,That Is the Question for Optimal Plasmid Extraction

Many molecular biology applications require fast plasmid DNA extraction, spurring multiple studies on how to speed up the process. It is regularly instructed in standard laboratory protocols to plate out frozen glycerol bacterial stocks prior to bacteria incubation in liquid media and subsequent plasmid extraction, although the rationale for this is often unexplained (other than for the isolation of single colonies). Given the commonality and importance of this laboratory operation, such a practice is time-consuming and laborious. To study the impact of this practice and the alternative direct culturing method, we investigated the association between bacterial cell mass and its potential influence on plasmid yields from the 2 methods. Our results showed no difference with preplating for 7 out of 8 plasmid constructs used in the study, suggesting that direct glycerol recovery would not lead to poorer plasmid yields. The findings support the rationale for direct glycerol recovery for plasmid extraction, without the need of an intermediate preplating step.     

To Model or not to Model,That is no Longer the Question for Ecologists

Here, I argue that we should abandon the division between “field ecologists” and “modelers,” and embrace modeling and empirical research as two powerful and often complementary approaches in the toolbox of 21st century ecologists, to be deployed alone or in combination depending on the task at hand. As empirical research has the longer tradition in ecology, and modeling is the more recent addition to the methodological arsenal, I provide both practical and theoretical reasons for integrating modeling more deeply into ecosystem research. Empirical research has epistemological priority over modeling; however, that is, for models to realize their full potential, and for modelers to wield this power wisely, empirical research is of fundamental importance. Combining both methodological approaches or forming “super ties” with colleagues using different methods are promising pathways to creatively exploit the methodological possibilities resulting from increasing computing power. To improve the proficiency of the growing group of model users and ensure future innovation in model development, we need to increase the modeling literacy among ecology students. However, an improved training in modeling must not curtail education in basic ecological principles and field methods, as these skills form the foundation for building and applying models in ecology.     

CRISPR-Cas: To Take Up DNA or Not-That Is the Question

Landmark experiments in the 1920s showed that capsule switching is critical for Streptococcus pneumonia survival. Further studies demonstrated that capsule "transformation" occurs via DNA uptake. In this issue of Cell Host and Microbe, Bikard et?al. (2012) show that CRISPR-Cas systems inhibit DNA uptake, selecting for the outgrowth of CRISPR-defective pneumococci.     

To Clone or Not to Clone—That Is the Question A Review of the Book “Cloning,” A. McLaren,Ed., Strasbourg: Council of Europe, 2002 (Ethical Eye Series)

Russian Journal of Developmental Biology -     

Evidence That Chlorinated Auxin Is Restricted to the Fabaceae But Not to the Fabeae

Auxin is a pivotal plant hormone, usually occurring in the form of indole-3-acetic acid (IAA). However, in maturing pea (Pisum sativum) seeds, the level of the chlorinated auxin, 4-chloroindole-3-acetic acid (4-Cl-IAA), greatly exceeds that of IAA. A key issue is how plants produce halogenated compounds such as 4-Cl-IAA. To better understand this topic, we investigated the distribution of the chlorinated auxin. We show for the first time, to our knowledge, that 4-Cl-IAA is found in the seeds of Medicago truncatula, Melilotus indicus, and three species of Trifolium. Furthermore, we found no evidence that Pinus spp. synthesize 4-Cl-IAA in seeds, contrary to a previous report. The evidence indicates a single evolutionary origin of 4-Cl-IAA synthesis in the Fabaceae, which may provide an ideal model system to further investigate the action and activity of halogenating enzymes in plants.The chlorinated form of auxin, 4-chloroindole-3-acetic acid (4-Cl-IAA), is a highly active hormone that is thought to play a key role in early pericarp growth (Reinecke et al., 1995, 1999; Ozga et al., 2009). Exogenous 4-Cl-IAA, for example, has been shown to promote the pericarp elongation of deseeded pea (Pisum sativum) pods (Reinecke et al., 1999). Johnstone et al. (2005) reported that 4-Cl-IAA and bioactive GA (GA₃ or GA₁) act synergistically on pericarp growth when applied simultaneously, and a growth regulatory role has been proposed for 4-Cl-IAA through induction of GA biosynthesis and inhibition of ethylene action. In other species, e.g. tomato (Solanum lycopersicum), the nonchlorinated form of auxin, indole-3-acetic acid (IAA), also stimulates fruit growth via GAs (Serrani et al., 2008; Tang et al., 2015). The chlorinated auxin is mainly found in reproductive structures (Katayama et al., 1988), in which its levels often exceed those of the more widespread IAA (Tivendale et al., 2012). The chlorinated form is thought to be restricted to members of the leguminous tribe Fabeae (Reinecke 1999), which includes the genera Vicia, Pisum, Lathyrus, Lens, and Vavilovia (Schaefer et al., 2012). However, there is a curious exception: 4-Cl-IAA has been reported also from Scots pine (Pinus sylvestris; Ernstsen and Sandberg, 1986).We previously published evidence that most 4-Cl-IAA in maturing pea seeds is synthesized from 4-Cl-tryptophan (4-Cl-Trp) via 4-Cl-indole-3-pyruvic acid (Tivendale et al., 2012, 2014). 4-Cl-Trp has been identified in extracts from pea and broad bean (Vicia faba) seeds (Kettner et al., 1992; Manabe et al., 1999), but whether the precursors of Trp can be chlorinated is unknown.Virtually nothing is known about the enzymes that catalyze halogenation reactions in plants. In bacteria, fungi, and marine algae, there are six types of enzymes responsible for the addition of halogen atoms to organic molecules. These include heme haloperoxidases, vanadium-dependent haloperoxidases, mononuclear nonheme iron halogenases, flavin-dependent halogenases, S-adenosyl-l-Met-dependent chlorinases and fluorinases, and methyl halide transferases (Butler and Sandy, 2009; Wagner et al., 2009). However, in the genomes of angiosperms, the only type of halogenating enzyme that has been annotated are haloperoxidases, but very little is known about these enzymes. To further understand the activity and action of halogenating enzymes in plants, a comparative system is required.In this study, we investigated the distribution of 4-Cl-IAA and 4-Cl-Trp in the Fabaceae by monitoring these compounds in the seeds of representative species spanning the phylogeny of this family. Most of these species have not been previously tested for the presence of the chlorinated compounds. In addition, we reexamined the reported occurrence of 4-Cl-IAA outside the Fabaceae, namely in Scots pine; several other Pinus species were investigated here as well. We also examined the endogenous levels of 4-Cl-IAA in both vegetative tissues and seeds of broad bean to address the question of whether 4-Cl-IAA is largely restricted to seeds (Pless et al., 1984; Katayama et al., 1988).