Seamless editing of the chloroplast genome in plants

BACKGROUND: Gene editing technologies enable the precise insertion of favourable mutations and performance enhancing trait genes into chromosomes whilst excluding all excess DNA from modified genomes. The technology gives rise to a new class of biotech crops which is likely to have widespread applications in agriculture. Despite progress in the nucleus, the seamless insertions of point mutations and non-selectable foreign genes into the organelle genomes of crops have not been described. The chloroplast genome is an attractive target to improve photosynthesis and crop performance. Current chloroplast genome engineering technologies for introducing point mutations into native chloroplast genes leave DNA scars, such as the target sites for recombination enzymes. Seamless editing methods to modify chloroplast genes need to address reversal of site-directed point mutations by template mediated repair with the vast excess of wild type chloroplast genomes that are present early in the transformation process. RESULTS: Using tobacco, we developed an efficient two-step method to edit a chloroplast gene by replacing the wild type sequence with a transient intermediate. This was resolved to the final edited gene by recombination between imperfect direct repeats. Six out of 11 transplastomic plants isolated contained the desired intermediate and at the second step this was resolved to the edited chloroplast gene in five of six plants tested. Maintenance of a single base deletion mutation in an imperfect direct repeat of the native chloroplast rbcL gene showed the limited influence of biased repair back to the wild type sequence. The deletion caused a frameshift, which replaced the five C-terminal amino acids of the Rubisco large subunit with 16 alternative residues resulting in a ~30-fold reduction in its accumulation. We monitored the process in vivo by engineering an overlapping gusA gene downstream of the edited rbcL gene. Translational coupling between the overlapping rbcL and gusA genes resulted in relatively high GUS accumulation (~0.5 % of leaf protein). CONCLUSIONS: Editing chloroplast genomes using transient imperfect direct repeats provides an efficient method for introducing point mutations into chloroplast genes. Moreover, we describe the first synthetic operon allowing expression of a downstream overlapping gene by translational coupling in chloroplasts. Overlapping genes provide a new mechanism for co-ordinating the translation of foreign proteins in chloroplasts. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12870-016-0857-6) contains supplementary material, which is available to authorized users

Frequency shift on the potential-dependent surface-enhanced Raman scattering of pyridine: simplified models for metal and solvent effects

The electronic structure of adsorbates is altered when it interacts with a surface, modifying the properties of both entities and giving rise to interesting phenomena related to heterogeneous catalysis or molecular electronics. If such surface is a metallic substrate, the electrode potential can be used to tune this interaction. Potential-dependent Surface-Enhanced Raman Scattering (SERS) is a particularly useful technique to study the induced effects on the molecule when the metal-adsorbate surface complex is formed, as the observed frequency shifts of the vibrational modes can provide information about it. However, from the computational point of view, these systems are difficult to model, because the macroscopic metal cannot be modelled easily using quantum mechanics. As an approach, we propose a simple model using silver atomic wires with different size and charge bonded to the molecule (AgnPyq, n = 2,3,5,7 and q = 0 and ±1 for n even and odd, respectively) which has been developed by the group and provides a good description of the effect of the electrode potential on the chemical enhancement mechanism of SERS.1-3 Electronic calculations were performed using Density Functional Theory (DFT). In order to study the frequency shifts, solvent effects have been taken into account by using the Polarizable Continuum Model (PCM). We have used three different functionals (B3LYP, PW91 and M06HF) and two basis sets (LANL2DZ for all atoms and LANL2DZ for Ag and 6-31G(d) for C,N,H) and, in all cases, a good agreement is achieved in terms of amplitude and trend of the frequency shift for most of the vibrational modes, especially when solvent interactions are included. The method was extended to other metals and solvents giving results in agreement with the available experimental data.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Charge Transfer mechanism in the Surface Enhanced Raman Scattering of 2,2'-bipyridine recorded on a silver electrode

Nowadays, Surface Enhanced Raman Spectroscopy (SERS) has become a powerful technique to investigate the electronic structure of surface-molecule hybrid systems due to the huge enhancement of the Raman signal. It is established that the origin of this enhancement has two main contributions; the electromagnetic (EM), related to surface plasmons, and the chemical mechanism, due to resonant charge transfer (CT) processes between the adsorbate and the metal. With the aim to investigate the SERS-CT of bipyridine and to identify charge transfer process, the spectra were recorded on silver electrode by using three different wavelengths (473, 532 and 785 nm) in a range from 0.0 up to -1.4 V electrode potential. The electrode potential was modelled in the calculations with atomic silver wires of different size and charge attached to the BPy molecule (AgnBPyq, with q = 0 for n = 2 and q = ±1 for n = 3, 5, 7) and were computed with Density Functional Theory (DFT). Although BPy shows a trans conformation in solution, a cis conformation was chosen for its chelating properties. The results indicate that the intensification of the ~1550 cm-1 band at negative potentials is due the Franck-Condon factors related to the resonant CT process from the metal to the BPy molecule.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

An MS-CASPT2 Study of the Photodecomposition of 4- Methoxyphenyl Azide. Role of Internal Conversion and Intersystem Crossing

Aryl azides photochemistry is strongly dependent on the substituent relative position, as has been studied by time resolved resonant Raman (TR3) spectroscopy for 4-methoxyphenyl azide and its isomer 3-methoxyphenyl azide. When irradiated at 266 nm, the former results in 4,4’-dimethoxyazobenzene whereas the latter forms 1,2-didehydroazepine. It is proposed that the key step of the reactions is the formation of a nitrene derivative. Recently, it has been proposed by us that nitrenes might have a relevant role in the Surface-Enhanced Raman Scattering (SERS) of p-aminothiophenol, however, the molecular mechanism is not well known in neither of these cases. Therefore, we studied the photodecomposition of 4-methoxyphenyl azide using multiconfigurational self-consistent field methods (MC-SCF) with the CAS-SCF and MS-CASPT2 approximations and calculated the resonant Raman spectra of the relevant species using a multi-state version of Albrecht’s vibronic theory. The results propose that the reaction follows a two steps sequence after irradiation at 266 nm: an intersystem crossing 21A’/23A’’ which decays through a 21A’/21A’’ conical intersection producing molecular nitrogen and triplet 4-methoxyphenyl nitrene in its ground state.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

¿SON IMPORTANTES LOS DATOS PERSONALES?

SOLO VISION PROYECTABLE