High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments

Single bacterial cells, each expressing a different library variant, were compartmentalized in aqueous droplets of water-in-oil (w/o) emulsions, thus maintaining a linkage between a plasmid-borne gene, the encoded enzyme variant, and the fluorescent product this enzyme may generate. Conversion into a double, water-in-oil-in-water (w/o/w) emulsion enabled the sorting of these compartments by FACS, as well as the isolation of living bacteria cells and their enzyme-coding genes. We demonstrate the directed evolution of new enzyme variants by screening >10(7) serum paraoxonase (PON1) mutants, to yield 100-fold improvements in thiolactonase activity. In vitro compartmentalization (IVC) of single cells, each carrying >10(4) enzyme molecules, in a volume of <10 femtoliter (fl), enabled detection and selection despite the fast, spontaneous hydrolysis of the substrate, the very low initial thiolactonase activity of PON1, and the use of difusable fluorescent products.     

The vibration-rotation emission spectrum of gaseous HZnCl

Gaseous HZnCl has been synthesized for the first time in a high-temperature tube furnace with a dc discharge in a flowing mixture of pure HCl and Zn vapor. The vibration-rotation emission spectrum of HZnCl was recorded at high resolution using a Fourier transform spectrometer. The H-Zn stretching modes (nu(1)) of the H(64)Zn(35)Cl, H(66)Zn(35)Cl, H(68)Zn(35)Cl, and H(64)Zn(37)Cl species, as well as the 2nu(1)-nu(1) hot band of the most abundant isotopologue H(64)Zn(35)Cl, were observed near 1966 cm(-1). A least-squares fit was performed for each of the four observed isotopologues, and their spectroscopic constants were determined.     

First measurements of the HCFC-142b trend from atmospheric chemistry experiment (ACE) solar occultation spectra

The first measurement of the HCFC-142b (CH₃CClF₂) trend near the tropopause has been derived from volume mixing ratio (VMR) measurements at northern and southern hemisphere mid-latitudes for the 2004–2008 time period from spaceborne solar occultation observations recorded at 0.02 cm⁻¹ resolution with the ACE (atmospheric chemistry experiment) Fourier transform spectrometer. The HCFC-142b molecule is currently the third most abundant HCFC (hydrochlorofluorocarbon) in the atmosphere and ACE measurements over this time span show a continuous rise in its volume mixing ratio. Monthly average measurements at northern and southern hemisphere mid-latitudes have similar increase rates that are consistent with surface trend measurements for a similar time span. A mean northern hemisphere profile for the time span shows a near constant VMR at 8–20 km altitude range, consistent on average for the same time span with in situ results. The nearly constant vertical VMR profile also agrees with model predictions of a long lifetime in the lower atmosphere.     

Fourier transform spectroscopy of chemiluminescence from the AΠ-XΣ system of SrO

The A^′1Π-X¹Σ⁺ near infrared system of strontium oxide (SrO) was observed at high spectral resolution by measuring the chemiluminescence from a Broida flow reactor using a Fourier transform spectrometer. In total, 32 bands from , , were measured within the spectral region at a resolution of . Vibrational levels of the upper state were observed up to v_A^′=4, and more than 5600 rotational lines were assigned. Incorporating previously published high resolution data for the A¹Σ⁺-X¹Σ⁺ system, a global fit to both data sets yields improved Dunham constants for the ground state and for the lower vibrational levels (v_A^′=0, 1, and 2) of the A^′1Π state. Because perturbations arising from interactions with the b³Σ⁺ and A¹Σ⁺ states affect the higher vibrational levels of the A^′1Π state more strongly, levels v_A^′=3 and 4 were represented by effective band constants in the fits. RKR potentials for the X¹Σ⁺,A^′1Π, and b³Σ⁺ states have been generated utilizing all the available data, Franck-Condon factors have been calculated for the A^′1Π-X¹Σ⁺ system, and A^′1Π∼b³Σ⁺ and A^′1Π∼A¹Σ⁺ perturbations are discussed.     

Characterization of the Ground State of Br(2) by Laser-Induced Fluorescence Fourier Transform Spectroscopy of the B(3)Pi(0(+))(u)-X(1)Sigma(+)(g) System

The laser-induced fluorescence (LIF) spectrum of the B(3)Pi(0(+))(u)-X(1)Sigma(+)(g) system of Br(2) was recorded by Fourier transform spectroscopy (FTS). The LIF spectra were obtained by using continuous-wave dye laser excitation in the spectral region 16 800-18 000 cm(-1). About 1800 rotationally resolved lines were recorded in 96 fluorescence progressions, originating from the 10 相似文献   

Spectroscopic requirements for ACCURATE, a microwave and infrared-laser occultation satellite mission

The proposed satellite mission ACCURATE consists of a small constellation of satellites in low Earth orbit, combining microwave occultation for thermodynamic state profiling with infrared-laser occultation for greenhouse gas and line-of-sight wind profiling. The mission aims to detect six greenhouse gas molecules with four additional isotopologues (H₂O, CO₂, CH₄, N₂O, O₃, CO, ¹³CO₂, OC¹⁸O, HDO, and H₂¹⁸O) in the upper troposphere and lower stratosphere in the 4000-5000 cm⁻¹ spectral region. Greenhouse gas profiles will be retrieved to within 1-2% accuracy using a ‘differential’ method, requiring two spectral points for each species - one to sample the spectral line and the other nearby to sample the baseline.An estimation of retrieval errors for the ACCURATE mission reveals that errors in spectroscopic line parameters dominate all other error sources. Poor knowledge of the spectroscopy introduces systematic errors into the retrieved greenhouse gas profiles. Using a simple approach, it was shown that the best line parameters currently available are too large to allow retrievals of greenhouse gases to within the stated ACCURATE mission goals of 1% accuracy for CO₂ and 2% for all other species. Therefore, spectroscopic line parameters for targeted lines need to be improved before the ACCURATE mission can be launched. Requirements have been formulated in this direction, and laboratory experiments outlined that could meet these requirements.     

Emission spectrum of hot HDO below 4000 cm

Fourier transform emission spectra were recorded using a mixture of H₂O and D₂O at a temperature of 1500 °C. The spectra were recorded in three overlapping sections and cover the wavenumber range 1800-3932 cm⁻¹. This spectrum is analyzed together with a previously reported one spanning the 380-2190 cm⁻¹ range [Parekunnel et al., J. Mol. Spectrosc. 2001 (28) 101]. This analysis leads to 4409 newly assigned HDO emission lines. This work particularly extends data on the (200) and (120) states of HDO for which newly determined energy levels are presented.     

Comparative extreme ultraviolet emission measurements for lithium and tin laser plasmas

Detailed spectroscopic studies on extreme UV emission from laser plasmas using tin and lithium planar solid targets were completed. At 13.5 nm, the best conversion efficiency (CE) for lithium was found to be 2.2% at intensities near 7 x 10(10) W/cm(2). The highest CE measured for tin was near 5.0% at an intensity close to 1 x 10(11) W/cm(2).