Saeid Kadkhodaei

Primary recovery of a bacteriocin-like inhibitory substance derived from Pediococcus acidilactici Kp10 by an aqueous two-phase system

A polymer-salt aqueous two-phase system (ATPS) consisting of polyethylene-glycol (PEG) with sodium citrate was developed for direct recovery of a bacteriocin-like inhibitory substance (BLIS) from a culture of Pediococcus acidilactici Kp10. The influences of phase composition, tie-line length (TLL), volume ratio (VR), crude sample loading, pH and sodium chloride (NaCl) on the partition behaviour of BLIS was investigated. Under optimum conditions of ATPS, the purification of BLIS was achieved at 26.5% PEG (8000)/11% sodium citrate with a TLL of 46.38% (w/w), VR of 1.8, and 1.8% crude load at pH 7 without the presence of NaCl. BLIS from P. acidilactici Kp10 was successfully purified by the ATPS up to 8.43-fold with a yield of 81.18%. Given that the operation of ATPS is simple, environmentally friendly and cost-effective, as it requires only salts and PEG, it may have potential for industrial applications in the recovery of BLIS from fermentation broth.

Enhancement of BLIS production by Pediococcus acidilactici kp10 in optimized fermentation conditions using an artificial neural network

The present study was aimed at enhancing the production of bacteriocin-like inhibitory substance (BLIS) produced by Pediococcus acidilactici Kp10 through optimizing the fermentation parameters. M17 was chosen in preliminary study as a culture medium because BLIS production was nine times higher (1427.7 AU mL−1) compared to that produced by MRS (160 AU mL−1). The fermentation parameters such as temperature, inoculum size, buffer strength, concentration of tween 80 and agitation speed were screened using two level half-factorial design. BLIS production is influenced by three most significant factors identified as temperature, inoculum size and agitation speed, which were further optimized using an artificial neural network (ANN). ANN predicted that a maximum activity of 5262.64 AU mL−1 would be obtained at optimum conditions of 120 rpm, 3% and 28.5 °C. The observed BLIS activity at the predicted optimum levels of the tested variables in ANN was 5118.5 AU mL−1, which was close to the predicted BLIS activity. Increased BLIS activity in the final solution, which resulted from the optimized process, would reduce downstream steps such as concentrating the product during purification.

Multiple overlap extension PCR (MOE-PCR): an effective technical shortcut to high throughput synthetic biology

The current study describes multiple-overlap-extension PCR (MOE-PCR) as a simple and effective approach to assembling multiple DNA fragments with various sizes and features in a single in vitro reaction. In this research, 50 bp of homology in overlapping DNA fragments and a specific touchdown PCR program resulted in successful assembly of eight different DNA fragments using a single PCR protocol. The simplicity, speed, reliability and cost-effectiveness of MOE-PCR offers it as an attractive method for assembling and/or cloning single and multiple DNA fragments. Indeed, the method is a one-step approach that eliminates some of the routine steps such as ligation and complex enzymatic reactions as well as sequence limitations of the other methods. The applications of this relatively high fidelity method could be extended to the construction of chimeric recombinant sequences that can be widely used in metabolic engineering, functional analysis of genes and genetic elements, expression studies of multi-domain proteins, protein engineering and the most recent genome editing strategies which together with synthetic biology are revolutionizing the life sciences. We expect the technique to be used as a robust, reliable and fast method in synthetic biology.

Prediction and In Silico Identification of Novel B-Cells and T-Cells Epitopes in the S1-Spike Glycoprotein of M41 and CR88 (793/B) Infectious Bronchitis Virus Serotypes for Application in Peptide Vaccines

Bioinformatic analysis was used to predict antigenic B-cell and T-cell epitopes within the S1 glycoprotein of M41 and CR88 IBV strains. A conserved linear B-cell epitope peptide, YTSNETTDVTS175–185, was identified in M41 IBV strains while three such epitopes types namely, VSNASPNSGGVD279–290, HPKCNFRPENI328–338, and NETNNAGSVSDCTAGT54–69, were predicted in CR88 IBV strains. Analysis of MHCI binding peptides in M41 IBV strains revealed the presence of 15 antigenic peptides out of which 12 were highly conserved in 96–100% of the total M41 strains analysed. Interestingly three of these peptides, GGPITYKVM208, WFNSLSVSI356, and YLADAGLAI472, relatively had high antigenicity index (>1.0). On the other hand, 11 MHCI binding epitope peptides were identified in CR88 IBV strains. Of these, five peptides were found to be highly conserved with a range between 90% and 97%. However, WFNSLSVSL358, SYNISAASV88, and YNISAASVA89 peptides comparably showed high antigenicity scores (>1.0). Combination of antigenic B-cells and T-cells peptides that are conserved across many strains as approach to evoke humoral and CTL immune response will potentially lead to a broad-based vaccine that could reduce the challenges in using live attenuated vaccine technology in the control of IBV infection in poultry.

Recovery and partial purification of thermophilic β-xylosidase derived from recombinant Bacillus megaterium MS941 by aqueous two-phase system

ABSTRACT A polymer–salt-based aqueous two-phase system (ATPS) was developed for the effective extraction and purification of extracellular β-xylosidase from the fermentation broth of recombinant Bacillus megaterium MS941. The effect of molecular weight (MW) of polyethylene glycol (PEG), tie-line length (TLL), volume ratio (VR), crude loading and pH on the recovery performance was evaluated. Under the optimal extraction conditions, β-xylosidase was successfully purified up to 23-fold with a recovery yield of 99% in the bottom salt-rich phase at PEG 4,000/potassium phosphate ATPS comprising TLL of 41.8, VR of 2.3, crude loading (CL) of 30% (w/w) at pH 6.

Functional Analyses of PtROS1-RNAi in Poplars and Evaluation of Its Effect on DNA Methylation

DNA methylation occurs mostly at the C5 position of dinucleotide symmetric CpG sites in genomic DNA. A balance is maintained in the plant genome between DNA methylation mediated by RNA-directed DNA methylation (RdDM) and DNA demethylation mediated by the DEMETER (DME) protein family and REPRESSOR OF SILENCING (ROS1). We used double-stranded RNA (dsRNA) silencing to suppress ROS1 protein expression in ‘Nanlin895’ (Populus deltoides × Populus euramericana ‘Nanlin895’). Leaves of WT and transformant poplars revealed more symmetric methylation on CpG sites than roots and stems. In addition, leaves of transformant poplars revealed more methylated CpG sites in both 5.8S rDNA and histone H3 compared to WT types via 0, 50 and 100 mM NaCl treatments. In asymmetric methylation sites, transformant poplars exhibited more methylated CpHpG and CpHpH contexts than WT poplars. On the other hand, hypermethylation induced by PtROS1-RNAi construct resulted in pleiotropic phenotypic changes in transgenic poplars. The percentage of wavy leaves was increased maximum by ~45% in transgenic poplars. Also, the number of leaves was increased by ~200 number in transformants. Furthermore, shooting (%) and rooting (%) was decreased in transgenic poplars versus WT.

Plant small RNAs: definition, classification and response against stresses

Gene knockdown and gene-silencing pathways in eukaryotic organisms are associated with small RNAs 20 to 25 nucleotides in length, which include microRNAs (miRNAs) and small interfering RNAs (siRNAs). These small RNAs are recruited to repress gene expression upstream or downstream of the transcription pathway. RNA interference (RNAi) is a biological inhibitor of gene expression that results in the destruction of messenger RNAs (mRNAs), leading to the inhibition of protein production. Indeed, RNA silencing plays a key role in plant development in terms of the plant’s response to both biotic and abiotic stresses. Conversely, Viral Suppressors of RNA silencing (VSRs) are proteins that hamper antiviral RNAi activation in plants, lead to suppress plant RNA-silencing. These VSR proteins prevent the induction of the plant antiviral RNAi immune response. This review focuses on small RNAs in plants and their roles in the responses of plants to biotic and abiotic stresses.

Cis/transgene optimization: systematic discovery of some key gene expression elements integrating bioinformatics and computational biology

In recombinant protein production, quantity and quality are the major challenges particularly for large scale and high-throughput production systems. The present study mainly focused on computational analysis and in silico systematic discovery of some key functional gene expression elements in microalgae Dunaliella salina as a case study which there is no or poor information in this regard. Among the key factors, we took a shot at matrix attachment regions (MARs), translation initiation sites (TIS), signal peptide (SP) sequences, gene optimization and transformation system. Computational analysis of MARs sequences provided enough information about the structure of these sequences and led us to design an artificial MAR sequence considering the essential motifs and underlain rules. As the consensus TIS, we revealed that A-3, G-6C-5C-4 and G+1C+2G+3 arrange the specific context in this microalgae which help in locating the ribosome at the correct reading frame. Bioinformatics studies unveiled the sequence of MASTRAPLLALLALLCAGSARA with the highest signal score as the specific SP for secretion systems. A multi-criteria optimization procedure was performed to redesign the coding sequence of the BAR selectable marker gene. The optimized version of the gene mainly covered the host codon preference, the less structured mRNA and exposure of TIS. As the intragenic factors, we selected an efficient promoter, a 5ˈ-UTR and an intron from the closely related species (Chlamydomonas Sp.) to construct the specific expression vectors. The expression cassettes containing optimized genetic elements could be delivered into the microalgae cells and conferred the resistance to the transformants for at least 90 generations. The findings indicated that the MARs flanking the expression cassette along with the optimized expression elements particularly codon adaptation could potentially improve transformation efficiency and stability. The findings can be efficiently deployed as an empirical model for systematic discovery of the key expression elements and optimization of the cis/transgenes.