Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler

Polymerase chain assembly (PCA) is a technique used to synthesize genes ranging from a few hundred base pairs to many kilobase pairs in length. In traditional PCA, equimolar concentrations of single stranded DNA oligonucleotides are repeatedly hybridized and extended by a polymerase enzyme into longer dsDNA constructs, with relatively few full-length sequences being assembled. Thus, traditional PCA is followed by a second primer-mediated PCR reaction to amplify the desired full-length sequence to useful, detectable quantities. Integration of assembly and primer-mediated amplification steps into a single reaction using a high-speed thermocycler is shown to produce similar results. For the integrated technique, the effects of oligo concentration, primer concentration, and number of oligonucleotides are explored. The technique is successfully demonstrated for the synthesis of two genes encoding EPCR-1 (653 bp) and pUC19 β-lactamase (929 bp) in under 20 min. However, rapid integrated PCA–PCR was found to be problematic when attempted with the TM-1 gene (1509 bp). Partial oligonucleotide sets of TM-1 could be assembled and amplified simultaneously, indicating that the technique may be limited to a maximum number of oligonucleotides due to competitive annealing and competition for primers

Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler

Polymerase chain assembly (PCA) is a technique used to synthesize genes ranging from a few hundred base pairs to many kilobase pairs in length. In traditional PCA, equimolar concentrations of single stranded DNA oligonucleotides are repeatedly hybridized and extended by a polymerase enzyme into longer dsDNA constructs, with relatively few full-length sequences being assembled. Thus, traditional PCA is followed by a second primer-mediated PCR reaction to amplify the desired full-length sequence to useful, detectable quantities. Integration of assembly and primer-mediated amplification steps into a single reaction using a high-speed thermocycler is shown to produce similar results. For the integrated technique, the effects of oligo concentration, primer concentration, and number of oligonucleotides are explored. The technique is successfully demonstrated for the synthesis of two genes encoding EPCR-1 (653 bp) and pUC19 β-lactamase (929 bp) in under 20 min. However, rapid integrated PCA–PCR was found to be problematic when attempted with the TM-1 gene (1509 bp). Partial oligonucleotide sets of TM-1 could be assembled and amplified simultaneously, indicating that the technique may be limited to a maximum number of oligonucleotides due to competitive annealing and competition for primers

Characterization of a Polymer Surface With Sequentially Immobilized Proteins

To overcome the procoagulant processes on the surfaces of biomaterials, surface modifications have been undertaken to achieve hemocompatability characteristics that are comparable to the native endothelium. Our immediate goal in this paper is to design and develop strategies to inhibit thrombin activation on biomaterial surfaces. We will use biodurable polyurethane (PU) as the background polymer and synthesize biomaterial surfaces containing two immobilized recombinant proteins. To attain our objective, we have first undertaken the surface modification of biodurable polyurethane (chronoflex- AR) to enable the sequential immobilization of proteins via a bi-dentate bridge, a novel modification strategy. We have verified the creation of the bridge by surface FT-IR conducted on each intermediate and the product of the synthesis. We estimate a yield of 0.25 μmol of the proposed bi-dentate bridge/cm2 polyurethane. We have characterized the protein binding on modified PU surfaces by immunofluorescence microscopy. As expected no visible fluorescence was detected on unmodified surfaces, while PU surfaces that has immobilized proteins via the bridge gave fluorescent signals, indicating the successful immobilization as per design. Results on surface modification and characterization of the resultant surface by FT-IR and dynamic mechanical analyses and immunofluorescence microscopy will be presented

Colour and AOX removal from pulping effluents by algae

A mixed culture of algae was used to treat pulping mill effluent in terms of removing both colour and adsorbably organic halides (AOX). The removal of AOX from pulping effluent increased with increasing initial colour Value of the effluent. However, for the total mill effluent (composed of both pulping and bleaching effluents), AOX removal was found to be independent of initial colour value, and was around 70%. Up to 80% removal of colour from pulping effluent was achieved within 30 days under continuous lighting conditions. It was found that algae reduced the colour of pulping effluent of relatively low initial colour more efficiently than that of high initial colour. Under simulated field lighting conditions, up to 60% colour removal from pulping, effluent was observed after 60 days of exposure, whereas for the total mill effluent it was up to 64% after 45 days. Total organic carbon and lignin (UVA(280)) were also removed to a significant extent, suggesting that the mechanism of colour removal might not be transformation of the coloured lignin molecules to non-coloured ones. Analysis of alkaline extraction of the algal biomass and material balance findings indicated that the main colour removal mechanism was metabolism rather than adsorption. The experimental results were also analysed using multiple regression techniques and a mathematical model was developed to express the removal of colour from pulping effluents in terms of initial colour value, exposure time and lighting periods as well as interactions between these variables

Algal treatment of pulp and paper industry wastewaters in SBR systems

Highly colored and highly polluted pulp and paper industry wastewaters are proposed to be treated by using algae in sequential batch reactors (SBR). Results of batch studies revealed that up to 74% COD; 74% color removal could be attained in about 40 days of incubation, From the preliminary SBR experiments, filling period was found to be a critical step affecting the overall efficiency when mixing and aeration is applied during filling. Therefore, 5 different filling periods (4, 6, 8, 10 and 12 days) were studied with a total SBR cycle of 15 days. For all filling periods; COD, color and AOX removal efficiencies increased with increasing filling time. Maximum removal efficiencies achieved were 60 to 85% for COD, 42 to 75% for color and 82 to 93% for AOX for the filling periods of 4 to 12 days. For 8 days or longer filling periods, no additional reaction time was required. Results showed that, organics in the wastewater were both chlorinated and non-chlorinated; algae removed these mainly by metabolism; and chlorine cleavage from chlorinated organic molecules was more rapid than the degradation of non-chlorinated and colored organics. Adsorbed lignin on algal biomass was found to be varying between 10-20% depending on filling period applied

Application of ozonation and biotreatment for forest industry wastewater

This research is focused on the integrated process for the treatment of bleached Kraft pulp mill effluents. Pre-ozonation of softwood and hardwood combined bleaching effluents at alkaline pH resulted in 50 and 44% COD abatement, respectively. Segregation of highly polluted streams of bleaching process can be recommended to reduce the cost of treatment since the COD removal yield of CEH effluents was higher than combined bleaching effluents. Moreover, biodegradability of CEH effluents in terms of BOD5/COD ratio was increased from 0.16 to 0.32. Noticeable color removal was achieved by pre-ozonation of the CEH stage and combined bleaching effluents. Pre-ozonation enhanced the performance of subsequent algal treatment in the sequential batch reactor (SBR) and activated sludge treatment. Algae have a higher ability to degrade AOX producing compounds together with a high COD removal rate. Although pre-ozonation increased the abatement rate of COD of SBR, the AOX removal rate remained constant as 87%

Merits of ozonation and catalytic ozonation pre-treatment in the algal treatment of pulp and paper mill effluents

Since the performance of algal treatment for pulp mill effluent decreases with increasing color intensity and AOX content, which mainly originate from the chlorine bleaching of Kraft pulp, the separated CEH bleaching effluent was pre-treated by both the conventional and the heterogeneous catalytic ozonation processes. An increase in the BOD5/COD, ratio from 0.11 to 0.28 and 87% color abatement in terms of Pt-Co were achieved by catalytic ozonation, which had the best treatment performance. Biodegradability enhancement of the CEH effluent correlated well with a decrease in toxicity, high-molecular-weight-compound content, and AOX abatement. By the pre-treatment of the CEH bleaching effluent, the overall efficiencies of algal treatment of the combined pulp mill effluent in terms of the fractional removal of COD and color were increased from 76% and 53% to 86-90% and 96-99%, respectively. Effects of both the conventional and the catalytic ozonation pre-treatments on subsequent biological treatment were close to each other and they reduced the filling period of the Sequential Batch Reactor (SBR) cycle from 8 to 5 days

New therapeutic system based on hydrogels for vaginal candidiasis management: formulation–characterization and in vitro evaluation based on vaginal irritation and direct contact test

PubMed: 327877182-s2.0-85089908135The objective of the present research was to examine the possible usage of terbinafine loaded hydrogels for vaginal application as part of vaginal candidiasis treatment. Vaginal candidiasis belongs to the most frequent gynecological disorders. Various antifungal drugs are used for its treatment, with Terbinafine being one of them. In this study, new gel formulations were prepared for Terbinafine vaginal delivery. Natural polymers such as chitosan, sodium carboxymethylcellulose, and Carbopol were used for the development of Terbinafine vaginal gels. The developed gels were examined for their viscosity and spreadability, pH and mechanical properties. The most optimal formulations were further evaluated for their in vitro release behavior and antifungal activities. In further, the cytotoxicity and irritation inducing capacity of optimum gel formulations were evaluated. In vitro drug release studies demonstrated that terbinafine release was prolonged whereas anti-candida activity in several species showed the superiority of the gels compared to the marketed product. G-5 and G-8 gels did not cause lysis, hemorrhage and coagulation, therefore, classified as non-irritant. The optimal formulations were also studied for their stability, demonstrating that they were stable for 3 months. © 2020 Informa UK Limited, trading as Taylor & Francis Group