Initiation of Protein Synthesis with Non-Canonical Amino Acids In Vivo

By transplanting identity elements into E. coli tRNA^fMet, we have engineered an orthogonal initiator tRNA (itRNA^Ty2) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNA^Ty2 can initiate translation in vivo with aromatic non-canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNA^fMet from the genome afforded an E. coli strain in which the efficiency of non-canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.     

Initiation of Protein Synthesis with Non‐Canonical Amino Acids In Vivo

By transplanting identity elements into E. coli tRNA^fMet, we have engineered an orthogonal initiator tRNA (itRNA^Ty2) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNA^Ty2 can initiate translation in vivo with aromatic non‐canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNA^fMet from the genome afforded an E. coli strain in which the efficiency of non‐canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.     

Selective Enzymatic Oxidation of Silanes to Silanols

Compared to the biological world's rich chemistry for functionalizing carbon, enzymatic transformations of the heavier homologue silicon are rare. We report that a wild‐type cytochrome P450 monooxygenase (P450_BM3 from Bacillus megaterium, CYP102A1) has promiscuous activity for oxidation of hydrosilanes to give silanols. Directed evolution was applied to enhance this non‐native activity and create a highly efficient catalyst for selective silane oxidation under mild conditions with oxygen as the terminal oxidant. The evolved enzyme leaves C?H bonds present in the silane substrates untouched, and this biotransformation does not lead to disiloxane formation, a common problem in silanol syntheses. Computational studies reveal that catalysis proceeds through hydrogen atom abstraction followed by radical rebound, as observed in the native C?H hydroxylation mechanism of the P450 enzyme. This enzymatic silane oxidation extends nature's impressive catalytic repertoire.     

Relative effectiveness of pretreatments on performance of Rhizomucor miehei lipase in nonpolar reaction media

Enzymes can be used in nonpolar reaction media to modify waterinsoluble substrates. A variety of pretreatments, applied to the enzyme prior to introduction to the nonpolar media, can improve enzyme activity. However, the various pretreatments have not been studied using directly comparable conditions, nor have they been applied simultaneously to test for interactive effects. This work evaluates pretreatment of lipase with various classes of additives. The pretreated lipase is used to catalyze esterification between citronellol and acetic acid in a medium of n-hexane. The effectiveness of a particular pretreatment is presented in terms of relative performance (RP), which is equal to the number of times faster the pretreated lipase catalyzes the reaction relative to untreated lipase. The individual and interactive effects of the pretreatment factors were studied and compared. Buffer salts had a much stronger performance-enhancing effect than nonbuffer salts; pretreatment with 90% (w/w) sodium phosphate yielded lipase with an RP of approx 64. A strong interaction was found between the treatments with sodium phosphate and pH adjustment. These treatments may mitigate the inhibitory effect of acetic acid. Activating effects of phase interfaces and active-site protectants are shown to be complementary to other treatments, demonstrating that they likely act by distinct mechanisms.     

Optimization of a New Extraction Technique for Analysis of Verbenone and cis-Verbenol in Pine Seeds

Results from a systematic study of the factors affecting extraction of cis-verbenol and verbenone from pine seeds are presented. Five extraction conditions were investigated: extraction solvent, method of extraction, extraction temperature, volume of solvent, and the ratio of the mass of sample to the amount of extraction solvent. The resulting optimized method uses magnetic-stirring-assisted extraction of pine seeds (5 g) with ethyl acetate (75 mL) for 20 min, at room temperature. RSDs were less than 5% for both compounds. GC–FID was used for quantification of cis-verbenol and verbenone in the extracts.     

Magnetic nanotips: the realisation of an atomic-scale beam-splitter

We present the first realisation of ferromagnetic Fe nanotips. Studies of the field emitted beams of electrons and metallic ions above and under the Curie temperature T_c were made. We have observed a reversible splitting of the electron beams when the temperature crossed through T_c and multiple spot patterns for the emitted metallic ion beams. These phenomena were not observed with Fe microtips or non-magnetic nanotips and thus were correlated to a strong magnetic interaction at the atomic scale apex of the nanotips. The Fe nanotips constitute an atomic scale beam-splitter.     

Bright‐Dark Rogue Waves

During the last two decades, revealing mechanisms of origin waves with anomalous amplitude (rogue waves) have been in the focus of researchers from different fields ranging from oceanography to laser physics. Mode‐locked lasers, as a test bed system, provide a unique opportunity to collect more data on rogue waves in the form of random pulses (soliton rain) and to clarify the mechanisms of rogue‐wave emergence caused by soliton–soliton and soliton–dispersive wave interactions. Here, for the first time, for an Er‐doped mode‐locked laser, a new type of vector rogue waves is demonstrated experimentally and theoretically, which is driven by desynchronization of the orthogonal linear states of polarization, so leading to output power oscillations in the form of anomalous spikes‐dips (bright‐dark rogue waves). The results can pave the way to unlocking the universal nature of the origin of rogue waves and thus can be of interest to the broad scientific community.     

Crosstalk between two-photon and two-color (two-photon) excitation in optical beam induced current generation with two confocal excitation beams

We analyze the influence of residual two-photon excitation (2PE) in two-color (two-photon) optical beam induced current (2CE-OBIC) generation in wide band gap semiconductor samples. 2CE-OBIC generation is accomplished with two confocal excitation beams of separation angle θ and wavelengths λ₁ and λ₂ where , λ_e = hc/E_b, h is the Planck’s constant, c is speed of light in vacuum, and E_b is the energy band gap. Because the conduction band of the sample is a continuum, at least one excitation beam would also contribute an undesirable 2PE-OBIC signal that degrades the signal-to-noise ratio of the measured 2CE-OBIC response and broadens the effective OBIC distribution in the sample particularly when θ ≠ 0 or π. We show that the deleterious effects of crosstalk are reduced by a careful selection of λ₁ and λ₂ and the relative excitation beam intensities. λ₁ and λ₂ should be chosen to minimize the ratio of the two-photon absorption coefficients (β₁, β₂) to the 2CE absorption coefficient β₁₂ or at least satisfy the constraint: β₁ + β₂  β₁₂. Keeping the two excitation intensities equal is beneficial only when β₁ = β₂. Otherwise, it is advantageous to bias the intensity ratio towards the wavelength with a lower 2PE absorption coefficient.