Lambda-Red recombineering is the most commonly used method to create point mutations, insertions or deletions in
and other bacteria, but usually an Flp recognition target (FRT) scar-site is retained ...in the genome. Alternative scarless recombineering methods, including CRISPR/Cas9-assisted methods, generally require cloning steps and/or complex PCR schemes for specific targeting of the genome. Here we describe the deletion of FRT scar-sites by the scarless Cas9-assisted recombineering method no-SCAR using an FRT-specific guide RNA, sgRNA
, and locus-specific ssDNA oligonucleotides. We applied this method to construct a scarless
strain suitable for gradual induction by l-arabinose. Genome sequencing of the resulting strain and its parent strains demonstrated that no additional mutations were introduced along with the simultaneous deletion of two FRT scar-sites. The FRT-specific no-SCAR selection by sgRNA
/Cas9 may be generally applicable to cure FRT scar-sites of
strains constructed by classical λ-Red recombineering.
Beta-cell-specific transgenic mice provide an invaluable model for dissecting the direct signaling mechanisms involved in regulating beta-cell structure and function. Furthermore, generating novel ...transgenic models is now easier and more cost-effective than ever, thanks to exciting novel approaches such as CRISPR.Here, we describe the commonly used approaches for generating and maintaining beta-cell-specific transgenic models and some of the considerations involved in their use. This includes the use of different beta-cell-specific promoters (e.g., pancreatic and duodenal homeobox factor 1 (Pdx1), rat insulin 2 promoter (RIP), and mouse insulin 1 promoter (MIP)) to drive site-specific recombinase technology. Important considerations during selection include level and uniformity of expression in the beta-cell population, ectopic transgene expression, and the use of inducible models.This chapter provides a guide to the procurement, generation, and maintenance of a beta-cell-specific transgene colony from preexisting Cre and loxP mouse strains, providing methods for crossbreeding and genotyping, as well as subsequent maintenance and, in the case of inducible models, transgenic induction.
A good catalyst for semihydrogenation of alkynes must preclude both over-hydrogenation of alkene to alkane and isomerization to the other alkene isomer. In addition, it should balance the trade-off ...between selectivity and activity. In 2013, the Repo and Pápai groups reported a frustrated Lewis pair (FLP) (1-NMe2-2-B(C6F5)2-C6H4), 1, which is a metal-free catalyst and for the first time shows excellent reactivity for the hydrogenation of internal alkynes. However, it is unreactive for terminal alkynes. In this work, we have designed 13 FLPs, a–m, based on 1 by varying the Lewis base site with N and P and the Lewis acid site with B, Al, Ga, and In and replacing pentafluorophenyl with 1,3,5-trifluorophenyl, phenyl, or trifluoromethyl. We apply density functional theory to study the activity, selectivity, and deactivation of FLP 1-m for acetylene semihydrogenation. The catalytic cycle consists of three steps: (1) alkyne insertion, (2) H2 heterolysis, and (3) intramolecular protonation. We found the activity does not change much by the modification of bulky ligands, while it decreases with the direct replacement of LA and LB sites. The overall activity depends on steps 1 and 3, which are, respectively, positively and negatively linear correlated with the charge of the Lewis acid site. Most of the FLPs in this work show comparable or better selectivity for semihydrogenation of acetylene than 1. FLP deactivation is due to the strong binding of acetylene and the elimination of electron-withdrawing bulky ligands at the preactivated catalyst rather than at activated catalysts. Taking the selectivity and stability of FLPs into account, we predict d and k are potentially active for terminal alkynes.
The Cre/loxP system is a versatile and powerful tool that has been used to develop many kinds of genetically modified mice, such as conditional knockout mice and mutant protein-expressing mice ...through the excision of a STOP cassette. However, while numerous in vivo and in vitro applications of the Cre/loxP system have been reported, it remains difficult to target at one time more than one set of recognition sites in an identical single cell in mice using the Cre/loxP system. To overcome this barrier, we developed two novel site-specific recombination systems called VCre/VloxP and SCre/SloxP. These systems allow multiple independent site-specific recombination, for example, multiple targeted deletions in the same cell at different times. In this chapter, I describe the features of VCre/VloxP and SCre/SloxP, practical protocols and tips on how to use them in genomic engineering applications, potential problems in their use, and how problems can be identified and solved.
Synthesizing azoles from ortho‐substituted anilines, CO2 and H2 has proved difficult due to the low nucleophilicity of anilines, which hinders their N‐formylation, i. e., the first step of the ...reaction. This study demonstrates that R3SnX Lewis acids (LA), N‐methylmorpholine (NMM) or DBU and polyethyleneimine (PEI) or N‐formylmorpholine efficiently catalyse the synthesis of benzimidazole and other azoles from ortho‐substituted anilines, CO2 and H2 by reductive coupling of CO2 to nucleophilic amine‐based solvents, PEI or morpholine, followed by in‐situ transfer of the formyl group to the appropriate ortho‐substituted aniline. Under these reaction conditions, spontaneous cyclization of the N‐formylated intermediate yields the corresponding azole in up to 98 % yield.
Sn‐based FLPs catalyse the synthesis of azoles by N‐formylating aliphatic amines with CO2 and H2 followed by in situ formate transfer to ortho‐substituted anilines and recycling of the aliphatic amine. This strategy eliminates issues with low reactivity of anilines in N‐formylations with CO2 and H2 and allows simultaneous recycling of the transfer formylation reagent.
The functionalization and reduction of CO2 are important targets not only because of concerns about its accumulation or the impact of this greenhouse gas on Earth’s climate, but also because of its ...potential as a C‐1 feedstock. In this work, both functionalization and reduction of CO2 were performed using tBu3P/ZnR2 (R=Et, I) “frustrated Lewis pairs” (FLPs). Phosphine‐catalyzed carboxylation of alkyl‐Zn bonds and catalytic reduction of CO2 using silanes as hydride sources and tBu3P/ ZnR2 (R=Et, I) FLPs as catalysts are reported. This is also the first time Zn‐based FLP chemistry in functionalization of CO2 is reported.
Artykuł prezentuje charakteryzację kobiet, które w Nowym Testamencie określone są explicite lub implicite jako głosicielki Ewangelii: (1) Maria Magdalena i inne kobiety przy grobie Jezusa; (2) ...kobieta samarytańska w J 4, 4–42; (3) Pryska/Pryscylla; (4) Ewodia i Syntyche z Flp 4, 2–3. Artykuł zwraca uwagę na historyczny i teologiczny kontekst ich działalności oraz definiuje treść i przedmiot ich głoszenia, czyli Ewangelię. W prezentacji każdej z kobiet lub grupy kobiet wskazano na najważniejsze problemy egzegetyczne i współczesne próby ich rozwiązania.
Ralstonia solanacearum
species complex (RSSC) is a group of Gram-negative bacterial pathogen capable of infecting numerous plants and crops, causing severe vascular wilt diseases. Functional analysis ...of the genes associated with bacterial virulence is critical for elucidating the molecular mechanisms that govern the bacterial pathogenicity. To this end, an efficient gene deletion method would be of great help. In this study, we set to develop an efficient and simple markerless gene deletion method by exploiting its natural transformation competence and the FLP/
FRT
recombination system. We found that natural transformation using PCR products provided much higher transformation frequency than the plasmid-based triparental mating and electroporation. We thus generated the gene deletion fusion PCR fragments by incorporating the upstream and downstream DNA fragments of the target gene and an antibiotic resistance gene flanked by
FRT
sites, and delivered the PCR products into
R. solanacearum
cells through natural transformation. Using this method, we knocked out the
epsB
and
phcA
genes, which are associated with exopolysaccharide (EPS) biosynthesis and regulation, respectively, in several
R. solanacearum
strains isolated from different host plants at a frequency from 5 (1E-08) to 45 (1E-08). To remove the antibiotic marker gene, the plasmid expressing the FLP enzyme was introduced into the above knockout mutants, which enabled removal of the marker gene. The effective combination of natural transformation and the FLP/
FRT
recombination system thus offers a simple and efficient method for functional study of putative virulence genes and for elucidation of
R. solanacearum
pathogenic mechanisms.
Podocytes are differentiated post-mitotic cells that cannot replace themselves after injury. Glomerular parietal epithelial cells are proposed to be podocyte progenitors. To test whether a subset of ...parietal epithelial cells transdifferentiate to a podocyte fate, dual reporter PEC-rtTA|LC1|tdTomato|Nphs1-FLPo|FRT-EGFP mice, named PEC-PODO, were generated. Doxycycline administration permanently labeled parietal epithelial cells with tdTomato reporter (red), and upon doxycycline removal, the parietal epithelial cells (PECs) cannot label further. Despite the presence or absence of doxycycline, podocytes cannot label with tdTomato, but are constitutively labeled with an enhanced green fluorescent protein (EGFP) reporter (green). Only activation of the Nphs1-FLPo transgene by labeled parietal epithelial cells can generate a yellow color. At day 28 of experimental focal segmental glomerulosclerosis, podocyte density was 20% lower in 20% of glomeruli. At day 56 of experimental focal segmental glomerulosclerosis, podocyte density was 18% lower in 17% of glomeruli. TdTomato+ parietal epithelial cells were restricted to Bowman’s capsule in healthy mice. However, by days 28 and 56 of experimental disease, two-thirds of tdTomato+ parietal epithelial cells within glomerular tufts were yellow in color. These cells co-expressed the podocyte markers podocin, nephrin, p57 and VEGF164, but not markers of endothelial (ERG) or mesangial (Perlecan) cells. Expansion microscopy showed primary, secondary and minor processes in tdTomato+EGFP+ cells in glomerular tufts. Thus, our studies provide strong evidence that parietal epithelial cells serve as a source of new podocytes in adult mice.
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•Unequal-area capability-based facility layout design approach is firstly introduced,•A novel mixed-integer non-linear programming (MINLP) model is developed,•Heuristic decomposition-based iterative ...mathematical programming method is proposed,•Application study and comparisons with a real-life manufacturing system.
Thus far in the available literature, capability-based facility layout design approaches were developed by only considering equal machine areas, where they are assigned to the pre-specified locations. Based on this motivation, this paper introduces a new unequal-area capability-based facility layout design (UA-CBFLD) problem for the first time in the literature. In addition to the machines’ unequal-area requirements, the proper distribution of their processing capabilities over the shop floor is considered. First, a new mixed-integer non-linear programming (MINLP) model is developed. Then, a polyhedral inner-approximation method is applied to cope with non-linear area constraints. Due to its NP-hard nature and highly non-linear structure, a heuristic decomposition-based iterative solution approach is also proposed to solve realistic size problems within a reasonable time. In order to show the validity and applicability of the proposed approach, both an illustrative example with different machine-capability overlaps and a real-life application in a manufacturing company are presented. The computational results have shown that the proposed approach has found a 33.25% better layout score (as a function of the distance and the amounts of product flows) and achieved this solution 116.64% faster than the solution of the developed MINLP model via Gurobi’s standard non-linear programming solver. Furthermore, when compared to the existing cellular facility layout design of the manufacturing company, the proposed capability-based unequal-area layout design option has also 29.37% and 34.74% superior layout scores under the scenarios of equal and unequal machine areas, respectively. Moreover, the total average machine utilization has increased up to 89.6%, which is comparatively better than the current average cell utilization, i.e., 65.2%.
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