A streamlined strategy for self-production of a commercial positive selection vector, the pJET1.2/blunt cloning vector, using common laboratory E. coli strains.

Positive selection vectors carry a lethal gene encoding a toxic product that is harmful to most laboratory E. coli strains. Previously, we reported a strategy for in-house production of a commercial positive selection vector, the pJET1.2/blunt cloning vector, using common laboratory E. coli strains. However, the strategy involves lengthy gel electrophoresis and extraction procedures to purify the linearized vector after digestion. Here, we streamlined the strategy to eliminate the gel-purification step. A uniquely designed short fragment called the Nawawi fragment was inserted into the coding sequence of the lethal gene of the pJET1.2 plasmid, resulting in the pJET1.2N plasmid that can be propagated in the E. coli strain DH5α. Digestion of the pJET1.2N plasmid with EcoRV released the Nawawi fragment, and the resulting blunt-ended pJET1.2/blunt cloning vector can be used directly for DNA cloning without prior purification. Cloning of a DNA fragment was not hindered by the Nawawi fragments carried over from the digestion step. After transformation, the pJET1.2N-derived pJET1.2/blunt cloning vector produced > 98% positive clones. The streamlined strategy accelerates the in-house production of the pJET1.2/blunt cloning vector and enables DNA cloning at a lower cost. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03647-3.

Reinvention of starch for oral drug delivery system design.

Starch is a polysaccharide with varying amylose-to-amylopectin ratios as a function of its biological sources. It is characterized by low shear stress resistance, poor aqueous/organic solubility and gastrointestinal digestibility which limit its ease of processing and functionality display as an oral drug delivery vehicle. Modulation of starch composition through genetic engineering primarily alters amylose-to-amylopectin ratio. Greater molecular properties changes require chemical and enzymatic modifications of starch. Acetylation reduces water solubility and enzymatic digestibility of starch. Carboxymethylation turns starch acid-insoluble and aggregative at low pHs. The summative effects are sustaining drug release in the upper gut. Acid-insoluble carboxymethylated starch can be aminated to provide an ionic character essential for hydrogel formation which further reduces its drug release. Ionic starch can coacervate with oppositely charged starch, non-starch polyelectrolyte or drug into insoluble, controlled-release complexes. Enzymatically debranched and resistant starch has a small molecular size which confers chain aggregation into a helical hydrogel network that traps the drug molecules, protecting them from biodegradation. The modified starch has been used to modulate the intestinal/colon-specific or controlled systemic delivery of oral small molecule drugs and macromolecular therapeutics. This review highlights synthesis aspects of starch and starch derivatives, and their outcomes and challenges of applications in oral drug delivery.

A strategy for in-house production of a positive selection cloning vector from the commercial pJET1.2/blunt cloning vector at minimal cost.

Key message: In-house production of a positive selection cloning vector could be simple, efficient and low cost. Abstract: DNA cloning technology requires a vector to harbour a gene of interest for multiplication of the gene in bacterial cells. Positive selection vector has become a popular type of cloning vector due to the simplicity and efficiency of the positive selection system. Due to the presence of a toxic gene, propagation of a commercial positive selection vector in common laboratory E. coli strains is infeasible. This study demonstrated a strategy for propagation and in-house production of a commercial positive selection vector, i.e., pJET1.2/blunt cloning vector, at low cost. This was done by insertion of a specially designed DNA fragment (MCS fragment), which can be easily removed later by EcoRV digestion, into the pJET1.2/blunt cloning vector to allow the propagation of the modified plasmid (termed pJET1.2M) in common E. coli strains. Removal of the MCS fragment from the pJET1.2M plasmid produces the pJET1.2/blunt cloning vector ready for gene cloning. The self-made pJET1.2/blunt cloning vector exhibited a cloning efficiency of 100%. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03289-x.

Genome-wide analysis of the CAD gene family reveals two bona fide CAD genes in oil palm.

Cinnamyl alcohol dehydrogenase (CAD) is the key enzyme for lignin biosynthesis in plants. In this study, genome-wide analysis was performed to identify CAD genes in oil palm (Elaeis guineensis). Phylogenetic analysis was then conducted to select the bona fide EgCADs. The bona fide EgCAD genes and their respective 5' flanking regions were cloned and analysed. Their expression profiles were evaluated in various organs using RT-PCR. Seven EgCAD genes (EgCAD1-7) were identified and divided into four phylogenetic groups. EgCAD1 and EgCAD2 display high sequence similarities with other bona fide CADs and possess all the signature motifs of the bona fide CAD. They also display similar 3D protein structures. Gene expression analysis showed that EgCAD1 was expressed most abundantly in the root tissues, while EgCAD2 was expressed constitutively in all the tissues studied. EgCAD1 possesses only one transcription start site, while EgCAD2 has five. Interestingly, a TC microsatellite was found in the 5' flanking region of EgCAD2. The 5' flanking regions of EgCAD1 and EgCAD2 contain lignin-associated regulatory elements i.e. AC-elements, and other defence-related motifs, including W-box, GT-1 motif and CGTCA-motif. Altogether, these results imply that EgCAD1 and EgCAD2 are bona fide CAD involved in lignin biosynthesis during the normal development of oil palm and in response to stresses. Our findings shed some light on the roles of the bona fide CAD genes in oil palm and pave the way for manipulating lignin content in oil palm through a genetic approach. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03208-0.

Characterization of promoter of EgPAL1, a novel PAL gene from the oil palm Elaeis guineensis Jacq.

KEY MESSAGE: The oil palm EgPAL1 gene promoter and its regulatory region were functional as a promoter in the heterologous system of Arabidopsis according to the cis-acting elements present in that region. The promoter was developmentally regulated, vascular tissue specific and responsive to water stress agents. Phenylalanine ammonia lyase (PAL, EC 4.3.1.24) is the key enzyme of the phenylpropanoid pathway which plays important roles in plant development and adaptation. To date, there is no report on the study of PAL from oil palm (Elaeis guineensis), an economically important oil crop. In this study, the 5' regulatory sequence of a highly divergent oil palm PAL gene (EgPAL1) was isolated and fused with GUS in Arabidopsis to create two transgenic plants carrying the minimal promoter with (2302 bp) and without its regulatory elements (139 bp). The regulatory sequence contained cis-acting elements known to be important for plant development and stress response including the AC-II element for lignin biosynthesis and several stress responsive elements. The promoter and its regulatory region were fully functional in Arabidopsis. Its activities were characterised by two common fundamental features of PAL which are responsive to plant internal developmental programme and external factors. The promoter was developmentally regulated in certain organs; highly active in young organs but less active or inactive in mature organs. The presence of the AC elements and global activity of the EgPAL1 promoter in all organs resembled the property of lignin-related genes. The existence of the MBS element and enhancement of the promoter activity by PEG reflected the behaviour of drought-responsive genes. Our findings provide a platform for evaluating oil palm gene promoters in the heterologous system of Arabidopsis and give insights into the activities of EgPAL1 promoter in oil palm.