Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3' Terminal Structures for Enzymatic De Novo DNA Synthesis.

Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3' terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin

CRISPR/Cas9 advances engineering of microbial cell factories

One of the key drivers for successful metabolic engineering in microbes is the efficacy by which genomes can be edited. As such there are many methods to choose from when aiming to modify genomes, especially those of model organisms like yeast and bacteria. In recent years, clustered regularly interspaced palindromic repeats (CRISPR) and its associated proteins (Cas) have become the method of choice for precision genome engineering in many organisms due to their orthogonality, versatility and efficacy. Here we review the strategies adopted for implementation of RNA-guided CRISPR/Cas9 genome editing with special emphasis on their application for metabolic engineering of yeast and bacteria. Also, examples of how nuclease-deficient Cas9 has been applied for RNA-guided transcriptional regulation of target genes will be reviewed, as well as tools available for computer-aided design of guide-RNAs will be highlighted. Finally, this review will provide a perspective on the immediate challenges and opportunities foreseen by the use of CRISPR/Cas9 genome engineering and regulation in the context of metabolic engineering

Selecting RNA aptamers for synthetic biology: investigating magnesium dependence and predicting binding affinity.

The ability to generate RNA aptamers for synthetic biology using in vitro selection depends on the informational complexity (IC) needed to specify functional structures that bind target ligands with desired affinities in physiological concentrations of magnesium. We investigate how selection for high-affinity aptamers is constrained by chemical properties of the ligand and the need to bind in low magnesium. We select two sets of RNA aptamers that bind planar ligands with dissociation constants (K(d)s) ranging from 65 nM to 100 microM in physiological buffer conditions. Aptamers selected to bind the non-proteinogenic amino acid, p-amino phenylalanine (pAF), are larger and more informationally complex (i.e., rarer in a pool of random sequences) than aptamers selected to bind a larger fluorescent dye, tetramethylrhodamine (TMR). Interestingly, tighter binding aptamers show less dependence on magnesium than weaker-binding aptamers. Thus, selection for high-affinity binding may automatically lead to structures that are functional in physiological conditions (1-2.5 mM Mg(2+)). We hypothesize that selection for high-affinity binding in physiological conditions is primarily constrained by ligand characteristics such as molecular weight (MW) and the number of rotatable bonds. We suggest that it may be possible to estimate aptamer-ligand affinities and predict whether a particular aptamer-based design goal is achievable before performing the selection

Global analysis of host response to induction of a latent bacteriophage

<p>Abstract</p> <p>Background</p> <p>The transition from viral latency to lytic growth involves complex interactions among host and viral factors, and the extent to which host physiology is buffered from the virus during induction of lysis is not known. A reasonable hypothesis is that the virus should be evolutionarily selected to ensure host health throughout induction to minimize its chance of reproductive failure. To address this question, we collected transcriptional profiles of <it>Escherichia coli </it>and bacteriophage lambda throughout lysogenic induction by UV light.</p> <p>Results</p> <p>We observed a temporally coordinated program of phage gene expression, with distinct early, middle and late transcriptional classes. Our study confirmed known host-phage interactions of induction of the heat shock regulon, escape replication, and suppression of genes involved in cell division and initiation of replication. We identified 728 <it>E. coli </it>genes responsive to prophage induction, which included pleiotropic stress response pathways, the Arc and Cpx regulons, and global regulators <it>crp </it>and <it>lrp</it>. Several hundred genes involved in central metabolism, energy metabolism, translation and transport were down-regulated late in induction. Though statistically significant, most of the changes in these genes were mild, with only 140 genes showing greater than two-fold change.</p> <p>Conclusion</p> <p>Overall, we observe that prophage induction has a surprisingly low impact on host physiology. This study provides the first global dynamic picture of how host processes respond to lambda phage induction.</p

Design and Construction of a Double Inversion Recombination Switch for Heritable Sequential Genetic Memory

Background: Inversion recombination elements present unique opportunities for computing and information encoding in biological systems. They provide distinct binary states that are encoded into the DNA sequence itself, allowing us to overcome limitations posed by other biological memory or logic gate systems. Further, it is in theory possible to create complex sequential logics by careful positioning of recombinase recognition sites in the sequence. Methodology/Principal Findings: In this work, we describe the design and synthesis of an inversion switch using the fim and hin inversion recombination systems to create a heritable sequential memory switch. We have integrated the two inversion systems in an overlapping manner, creating a switch that can have multiple states. The switch is capable of transitioning from state to state in a manner analogous to a finite state machine, while encoding the state information into DNA. This switch does not require protein expression to maintain its state, and ‘‘remembers’ ’ its state even upon cell death. We were able to demonstrate transition into three out of the five possible states showing the feasibility of such a switch. Conclusions/Significance: We demonstrate that a heritable memory system that encodes its state into DNA is possible, and that inversion recombination system could be a starting point for more complex memory circuits. Although the circuit di