Displaying publications 1 - 20 of 60 in total

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  1. Enhancers of Agrobacterium-mediated Transformation of Tibouchina semidecandra Selected on the Basis of GFP Expression
    Yong WT, Henry ES, Abdullah JO
    Trop Life Sci Res, 2010 Dec;21(2):115-30.
    PMID: 24575204
    Genetic engineering is a powerful tool for the improvement of plant traits. Despite reported successes in the plant kingdom, this technology has barely scratched the surface of the Melastomataceae family. Limited studies have led to some optimisation of parameters known to affect the transformation efficiency of these plants. The major finding of this study was to optimise the presence of selected enhancers [e.g., monosaccharides (D-glucose, D-galactose and D-fructose), tyrosine, aluminium chloride (AICI3) and ascorbic acid] to improve the transformation efficiency of Tibouchina semidecandra. Agrobacterium tumefaciens strain LBA4404 harbouring the disarmed plasmid pCAMBIA1304 was used to transform shoots and nodes of T. semidecandra. Different concentrations of the transformation enhancers were tested by using green fluorescent protein (GFP) as a reporter. The results obtained were based on the percentage of GFP expression, which was observed 14 days post-transformation. A combination of 120 μM galactose and 100 μM tyrosine supplemented with 600 μM AICI3 in the presence of 15 mg/l ascorbic acid gave the highest percentage of positive transformants for T. semidecandra shoots. Whereas 60 μM galactose and 50 μM tyrosine with 200 μM AICI3 in the presence of 15 mg/l ascorbic acid was optimum for T. semidecandra nodes. The presence of the hygromycin phosphotransferase II (hptII) transgene in the genomic DNA of putative T. semidecandra transformants was verified by PCR amplification with specific primers.
    Matched MeSH terms: Genetic Engineering
  2. Fulltext Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications
    Yeo CC, Abu Bakar F, Chan WT, Espinosa M, Harikrishna JA
    Toxins (Basel), 2016 Feb 19;8(2):49.
    PMID: 26907343 DOI: 10.3390/toxins8020049
    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.
    Matched MeSH terms: Genetic Engineering
  3. Fulltext Elevated CO2 improves both lipid accumulation and growth rate in the glucose-6-phosphate dehydrogenase engineered Phaeodactylum tricornutum
    Wu S, Gu W, Huang A, Li Y, Kumar M, Lim PE, et al.
    Microb Cell Fact, 2019 Sep 23;18(1):161.
    PMID: 31547820 DOI: 10.1186/s12934-019-1214-x
    BACKGROUND: Numerous studies have shown that stress induction and genetic engineering can effectively increase lipid accumulation, but lead to a decrease of growth in the majority of microalgae. We previously found that elevated CO2 concentration increased lipid productivity as well as growth in Phaeodactylum tricornutum, along with an enhancement of the oxidative pentose phosphate pathway (OPPP) activity. The purpose of this work directed toward the verification of the critical role of glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme in the OPPP, in lipid accumulation in P. tricornutum and its simultaneous rapid growth rate under high-CO2 (0.15%) cultivation.

    RESULTS: In this study, G6PDH was identified as a target for algal strain improvement, wherein G6PDH gene was successfully overexpressed and antisense knockdown in P. tricornutum, and systematic comparisons of the photosynthesis performance, algal growth, lipid content, fatty acid profiles, NADPH production, G6PDH activity and transcriptional abundance were performed. The results showed that, due to the enhanced G6PDH activity, transcriptional abundance and NAPDH production, overexpression of G6PDH accompanied by high-CO2 cultivation resulted in a much higher of both lipid content and growth in P. tricornutum, while knockdown of G6PDH greatly decreased algal growth as well as lipid accumulation. In addition, the total proportions of saturated and unsaturated fatty acid, especially the polyunsaturated fatty acid eicosapentaenoic acid (EPA; C20:5, n-3), were highly increased in high-CO2 cultivated G6PDH overexpressed strains.

    CONCLUSIONS: The successful of overexpression and antisense knockdown of G6PDH well demonstrated the positive influence of G6PDH on algal growth and lipid accumulation in P. tricornutum. The improvement of algal growth, lipid content as well as polyunsaturated fatty acids in high-CO2 cultivated G6PDH overexpressed P. tricornutum suggested this G6PDH overexpression-high CO2 cultivation pattern provides an efficient and economical route for algal strain improvement to develop algal-based biodiesel production.

    Matched MeSH terms: Genetic Engineering
  4. Expression and characterization of a cellobiohydrolase (CBH7B) from the thermophilic fungus Thielavia terrestris in Pichia pastoris
    Woon JS, Mackeen MM, Mahadi NM, Illias RM, Abdul Murad AM, Abu Bakar FD
    Biotechnol Appl Biochem, 2016 Sep;63(5):690-698.
    PMID: 26265428 DOI: 10.1002/bab.1431
    The gene encoding a cellobiohydrolase 7B (CBH7B) of the thermophilic fungus Thielavia terrestris was identified, subcloned, and expressed in Pichia pastoris. CBH7B encoded 455 amino acid residues with a molecular mass of 51.8 kDa. Domain analysis indicated that CBH7B contains a family 7 glycosyl hydrolase catalytic core but lacks a carbohydrate-binding module. Purified CBH7B exhibited optimum catalytic activity at pH 5.0 and 55 °C with 4-methylumbelliferryl-cellobioside as the substrate and retained 85% of its activity following 24 H incubation at 50 °C. Despite the lack of activity toward microcrystalline substrates, this enzyme worked synergistically with the commercial enzyme cocktail Cellic(®) CTec2 to enhance saccharification by 39% when added to a reaction mixture containing 0.25% alkaline pretreated oil palm empty fruit bunch (OPEFB). Attenuated total reflectance Fourier transform infrared spectroscopy suggested a reduction of lignin and crystalline cellulose in OPEFB samples supplemented with CBH7B. Scanning electron microscopy revealed greater destruction extent of OPEFB strands in samples supplemented with CBH7B as compared with the nonsupplemented control. Therefore, CBH7B has the potential to complement commercial enzymes in hydrolyzing lignocellulosic biomass.
    Matched MeSH terms: Genetic Engineering/methods*
  5. Fulltext Engineering the production of major catechins by Escherichia coli carrying metabolite genes of Camellia sinensis
    Umar KM, Abdulkarim SM, Radu S, Abdul Hamid A, Saari N
    ScientificWorldJournal, 2012;2012:529031.
    PMID: 22645428 DOI: 10.1100/2012/529031
    A mimicked biosynthetic pathway of catechin metabolite genes from C. sinensis, consisting of flavanone 3 hydroxylase (F3H), dihydroflavonol reductase (DFR), and leucoanthocyanidin reductase (LCR), was designed and arranged in two sets of constructs: (a) single promoter in front of F3H and ribosome-binding sequences both in front of DFR and LCR; (b) three different promoters with each in the front of the three genes and ribosome-binding sequences at appropriate positions. Recombinant E. coli BL (DE3) harbouring the constructs were cultivated for 65 h at 26 °C in M9 medium consisting of 40 g/L glucose, 1 mM IPTG, and 3 mM eriodictyol. Compounds produced were extracted in ethyl acetate in alkaline conditions after 1 h at room temperature and identified by HPLC. Two of the four major catechins, namely, (-)-epicatechin (0.01) and (-)-epicatechin gallate (0.36 mg/L), and two other types ((+)-catechin hydrate (0.13 mg/L) and (-)-catechin gallate (0.04 mg/L)) were successfully produced.
    Matched MeSH terms: Genetic Engineering/methods
  6. Fulltext The Use of Genetic and Gene Technologies in Shaping Modern Rapeseed Cultivars (Brassica napus L.)
    Ton LB, Neik TX, Batley J
    Genes (Basel), 2020 09 30;11(10).
    PMID: 33008008 DOI: 10.3390/genes11101161
    Since their domestication, Brassica oilseed species have undergone progressive transformation allied with the development of breeding and molecular technologies. The canola (Brassica napus) crop has rapidly expanded globally in the last 30 years with intensive innovations in canola varieties, providing for a wider range of markets apart from the food industry. The breeding efforts of B. napus, the main source of canola oil and canola meal, have been mainly focused on improving seed yield, oil quality, and meal quality along with disease resistance, abiotic stress tolerance, and herbicide resistance. The revolution in genetics and gene technologies, including genetic mapping, molecular markers, genomic tools, and gene technology, especially gene editing tools, has allowed an understanding of the complex genetic makeup and gene functions in the major bioprocesses of the Brassicales, especially Brassica oil crops. Here, we provide an overview on the contributions of these technologies in improving the major traits of B. napus and discuss their potential use to accomplish new improvement targets.
    Matched MeSH terms: Genetic Engineering*
  7. Fulltext Enhancers of Agrobacterium-mediated transformation of Tibouchina semidecandra selected on the basis of GFP expression
    Thau, Wilson Lym Yon, Henry, Erle Stanley, Janna Ong Abdullah
    Trop Life Sci Res, 2010;21(2):-.
    MyJurnal
    Genetic engineering is a powerful tool for the improvement of plant traits. Despite reported successes in the plant kingdom, this technology has barely scratched the surface of the Melastomataceae family. Limited studies have led to some optimisation of parameters known to affect the transformation efficiency of these plants. The major finding of this study was to optimise the presence of selected enhancers [e.g., monosaccharides (D-glucose, D-galactose and D-fructose), tyrosine, aluminium chloride (AICI3) and ascorbic
    acid] to improve the transformation efficiency of Tibouchina semidecandra. Agrobacterium tumefaciens strain LBA4404 harbouring the disarmed plasmid pCAMBIA1304 was used to transform shoots and nodes of T. semidecandra. Different concentrations of the transformation enhancers were tested by using green fluorescent protein (GFP) as a reporter. The results obtained were based on the percentage of GFP expression, which was observed 14 days post-transformation. A combination of 120 µM galactose and 100
    µM tyrosine supplemented with 600 µM AICI3 in the presence of 15 mg/l ascorbic acid gave the highest percentage of positive transformants for T. semidecandra shoots. Whereas 60 µM galactose and 50 µM tyrosine with 200 µM AICI3 in the presence of 15 mg/l ascorbic acid was optimum for T. semidecandra nodes. The presence of the hygromycin phosphotransferase II (hptII) transgene in the genomic DNA of putative
    T. semidecandra transformants was verified by PCR amplification with specific primers.
    Matched MeSH terms: Genetic Engineering
  8. Fulltext Engineering the lactococcal mevalonate pathway for increased sesquiterpene production
    Song AA, Abdullah JO, Abdullah MP, Shafee N, Othman R, Noor NM, et al.
    FEMS Microbiol Lett, 2014 Jun;355(2):177-84.
    PMID: 24828482 DOI: 10.1111/1574-6968.12469
    Isoprenoids are a large, diverse group of secondary metabolites which has recently raised a renewed research interest due to genetic engineering advances, allowing specific isoprenoids to be produced and characterized in heterologous hosts. Many researches on metabolic engineering of heterologous hosts for increased isoprenoid production are focussed on Escherichia coli and yeasts. E. coli, as most prokaryotes, use the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway for isoprenoid production. Yeasts on the other hand, use the mevalonate pathway which is commonly found in eukaryotes. However, Lactococcus lactis is an attractive alternative host for heterologous isoprenoid production. Apart from being food-grade, this Gram-positive prokaryote uses the mevalonate pathway for isoprenoid production instead of the MEP pathway. Previous studies have shown that L. lactis is able to produce sesquiterpenes through heterologous expression of plant sesquiterpene synthases. In this work, we analysed the gene expression of the lactococcal mevalonate pathway through RT-qPCR to successfully engineer L. lactis as an efficient host for isoprenoid production. We then overexpressed the mvk gene singly or co-expressed with the mvaA gene as an attempt to increase β-sesquiphellandrene production in L. lactis. It was observed that co-expression of mvk with mvaA doubled the amount of β-sesquiphellandrene produced.
    Matched MeSH terms: Genetic Engineering/methods*
  9. Fulltext A review on Lactococcus lactis: from food to factory
    Song AA, In LLA, Lim SHE, Rahim RA
    Microb Cell Fact, 2017 04 04;16(1):55.
    PMID: 28376880 DOI: 10.1186/s12934-017-0669-x
    Lactococcus lactis has progressed a long way since its discovery and initial use in dairy product fermentation, to its present biotechnological applications in genetic engineering for the production of various recombinant proteins and metabolites that transcends the heterologous species barrier. Key desirable features of this gram-positive lactic acid non-colonizing gut bacteria include its generally recognized as safe (GRAS) status, probiotic properties, the absence of inclusion bodies and endotoxins, surface display and extracellular secretion technology, and a diverse selection of cloning and inducible expression vectors. This have made L. lactis a desirable and promising host on par with other well established model bacterial or yeast systems such as Escherichia coli, Saccharomyces [corrected] cerevisiae and Bacillus subtilis. In this article, we review recent technological advancements, challenges, future prospects and current diversified examples on the use of L. lactis as a microbial cell factory. Additionally, we will also highlight latest medical-based applications involving whole-cell L. lactis as a live delivery vector for the administration of therapeutics against both communicable and non-communicable diseases.
    Matched MeSH terms: Genetic Engineering/methods*
  10. Dissolving the engineering moral dilemmas within the Islamic ethico-legal praxes
    Solihu AK, Ambali AR
    Sci Eng Ethics, 2011 Mar;17(1):133-47.
    PMID: 19937149 DOI: 10.1007/s11948-009-9185-7
    The goal of responsible engineers is the creation of useful and safe technological products and commitment to public health, while respecting the autonomy of the clients and the public. Because engineers often face moral dilemma to resolve such issues, different engineers have chosen different course of actions depending on their respective moral value orientations. Islam provides a value-based mechanism rooted in the Maqasid al-Shari'ah (the objectives of Islamic law). This mechanism prioritizes some values over others and could help resolve the moral dilemmas faced in engineering. This paper introduces the Islamic interpretive-evaluative maxims to two core issues in engineering ethics: genetically modified foods and whistleblowing. The study aims primarily to provide problem-solving maxims within the Maqasid al-Shari'ah matrix through which such moral dilemmas in science and engineering could be studied and resolved.
    Matched MeSH terms: Genetic Engineering/ethics
  11. Fourth generation biofuel from genetically modified algal biomass: Challenges and future directions
    Shokravi H, Shokravi Z, Heidarrezaei M, Ong HC, Rahimian Koloor SS, Petrů M, et al.
    Chemosphere, 2021 Dec;285:131535.
    PMID: 34329137 DOI: 10.1016/j.chemosphere.2021.131535
    Genetic engineering applications in the field of biofuel are rapidly expanding due to their potential to boost biomass productivity while lowering its cost and enhancing its quality. Recently, fourth-generation biofuel (FGB), which is biofuel obtained from genetically modified (GM) algae biomass, has gained considerable attention from academic and industrial communities. However, replacing fossil resources with FGB is still beset with many challenges. Most notably, technical aspects of genetic modification operations need to be more fully articulated and elaborated. However, relatively little attention has been paid to GM algal biomass. There is a limited number of reviews on the progress and challenges faced in the algal genetics of FGB. Therefore, the present review aims to fill this gap in the literature by recapitulating the findings of recent studies and achievements on safe and efficient genetic manipulation in the production of FGB. Then, the essential issues and parameters related to genome editing in algal strains are highlighted. Finally, the main challenges to FGB pertaining to the diffusion risk and regulatory frameworks are addressed. This review concluded that the technical and biosafety aspects of FGB, as well as the complexity and diversity of the related regulations, legitimacy concerns, and health and environmental risks, are among the most important challenges that require a strong commitment at the national/international levels to reach a global consensus.
    Matched MeSH terms: Genetic Engineering
  12. Ethical issues of genetically modified foods (GMF)
    Shamsinar BA
    Med J Malaysia, 2000 Aug;55 Suppl B:62-4.
    PMID: 11125525
    Matched MeSH terms: Genetic Engineering*
  13. Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) by recombinant Escherichia coli expressing leucine metabolism-related enzymes derived from Clostridium difficile
    Saika A, Watanabe Y, Sudesh K, Tsuge T
    J Biosci Bioeng, 2014 Jun;117(6):670-5.
    PMID: 24484910 DOI: 10.1016/j.jbiosc.2013.12.006
    An obligate anaerobic bacterium Clostridium difficile has a unique metabolic pathway to convert leucine to 4-methylvalerate, in which 4-methyl-2-pentenoyl-CoA (4M2PE-CoA) is an intermediate of this pathway. 4M2PE-CoA is also able to be converted to 3-hydroxy-4-methylvalerate (3H4MV), a branched side chain monomer unit, for synthesis of polyhydroxyalkanoate (PHA) copolymer. In this study, to synthesize 3H4MV-containing PHA copolymer from leucine, the leucine metabolism-related enzymes (LdhA and HadAIBC) derived from C. difficile and PHA biosynthesis enzymes (PhaPCJAc and PhaABRe) derived from Aeromonas caviae and Ralstonia eutropha were co-expressed in the codon usage-improved Escherichia coli. Under microaerobic culture conditions, this E. coli was able to synthesize P(3HB-co-12.2 mol% 3H4MV) from glucose with the supplementation of 1 g/L leucine. This strain also produced P(3HB-co-12.6 mol% 3H4MV) using the culture supernatant of leucine overproducer E. coli strain NS1391 as the medium for PHA production, achieving 3H4MV copolymer synthesis only from glucose. Furthermore, we tested the feasibility of the 3H4MV copolymer synthesis in E. coli strain NS1391 from glucose. The recombinant E. coli NS1391 was able to synthesize P(3HB-co-3.0 mol% 3H4MV) from glucose without any leucine supplementation. This study demonstrates the potential of the new metabolic pathway for 3H4MV synthesis using leucine metabolism-related enzymes from C. difficile.
    Matched MeSH terms: Genetic Engineering
  14. Insulin secreted from genetically engineered intestinal cells reduces blood glucose levels in diabetic mice
    Rasouli M, Allaudin ZN, Omar AR, Ahmad Z
    Curr Gene Ther, 2013 Aug;13(4):229-39.
    PMID: 23721205 DOI: 10.2174/15665232113139990002
    Poorly controlled diabetes mellitus can result in serious complications. Gene therapy is increasingly being considered as an alternative approach to treat diabetes, because of its ability to induce physiological insulin secretion and it allows patients to escape insulin injections. The properties of gut K and L-cells, including glucose sensitivity, the ability to process insulin and a regulated secretion pathway support their use as surrogate β-cells. Previous in vitro studies have provided sufficient evidence supporting the use of these cells for gene therapy studies. Therefore, we examined the ability of K and L-cells to produce insulin in diabetic mice. Chitosan nanoparticles were used to transfer the insulin gene into intestinal cells via oral administration. The efficiency of chitosan as a gene vehicle was investigated through the use of reporter gene. Insulin mRNA and protein expression levels were measured by RT-PCR and ELISA, respectively. Blood glucose testing revealed that this treatment reduced glucose levels in diabetic mice. The decrease in blood glucose level in the first week of treatment was greater in mice with K-cell specific insulin expression compared with mice with L-cell-specific insulin expression. These results indicate that inducing insulin secretion in K-cells conferred a quicker response to gene therapy.
    Matched MeSH terms: Genetic Engineering*
  15. Fulltext Engineering an L-cell line that expresses insulin under the control of the glucagon-like peptide-1 promoter for diabetes treatment
    Rasouli M, Ahmad Z, Omar AR, Allaudin ZN
    BMC Biotechnol, 2011 Nov 03;11:99.
    PMID: 22047106 DOI: 10.1186/1472-6750-11-99
    BACKGROUND: Diabetes mellitus is a complicated disease with a pathophysiology that includes hyperinsulinemia, hyperglycemia and other metabolic impairments leading to many clinical complications. It is necessary to develop appropriate treatments to manage the disease and reduce possible acute and chronic side effects. The advent of gene therapy has generated excitement in the medical world for the possible application of gene therapy in the treatment of diabetes. The glucagon-like peptide-1 (GLP-1) promoter, which is recognised by gut L-cells, is an appealing candidate for gene therapy purposes. The specific properties of L-cells suggest that L-cells and the GLP-1 promoter would be useful for diabetes therapy approaches.

    RESULTS: In this study, L-cells were isolated from a primary intestinal cell line to create suitable target cells for insulin expression studies. The isolated cells displayed L-cell properties and were therefore used as an L-cell surrogate. Next, the isolated L-cells were transfected with the recombinant plasmid consisting of an insulin gene located downstream of the GLP-1 promoter. The secretion tests revealed that an increase in glucose concentration from 5 mM to 25 mM induced insulin gene expression in the L-cells by 2.7-fold. Furthermore, L-cells quickly responded to the glucose stimulation; the amount of insulin protein increased 2-fold in the first 30 minutes and then reached a plateau after 90 minutes.

    CONCLUSION: Our data showed that L-cells efficiently produced the mature insulin protein. In addition, the insulin protein secretion was positively regulated with glucose induction. In conclusion, GLP-1 promoter and L-cell could be potential candidates for diabetes gene therapy agents.

    Matched MeSH terms: Genetic Engineering/methods*
  16. Achieving crop stress tolerance and improvement--an overview of genomic techniques
    Rasool S, Ahmad P, Rehman MU, Arif A, Anjum NA
    Appl Biochem Biotechnol, 2015 Dec;177(7):1395-408.
    PMID: 26440315 DOI: 10.1007/s12010-015-1830-9
    The inexorable exposure of plants to the combinations of abiotic stresses has affected the worldwide food supply. The crop improvement against these abiotic stresses has been captivating approach to increase the yield and enhance the stress tolerance. By using traditional and modern breeding methods, the characters that confer tolerance to these stresses were accomplished. No doubt genetic engineering and molecular breeding have helped in comprehending the intricate nature of stress response. Understanding of abiotic stress-involved cellular pathways provides vital information on such responses. On the other hand, genomic research for crop improvement has raised new assessments in breeding new varieties against abiotic stresses. Interpretation of responses of the crop plants under stress is of great significance by studying the main role of crops in food and biofuel production. This review presents genomic-based approaches revealing the complex networks controlling the mechanisms of abiotic stress tolerance, and the possible modes of assimilating information attained by genomic-based approaches due to the advancement in isolation and functional analysis of genes controlling the yield and abiotic stress tolerance are discussed.
    Matched MeSH terms: Genetic Engineering
  17. Characterization of Chlamydomonas Ribulose-1,5-bisphosphate carboxylase/oxygenase variants mutated at residues that are post-translationally modified
    Rasineni GK, Loh PC, Lim BH
    Biochim Biophys Acta Gen Subj, 2017 Feb;1861(2):79-85.
    PMID: 27816753 DOI: 10.1016/j.bbagen.2016.10.027
    BACKGROUND: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the chloroplast enzyme that fixes CO2 in photosynthesis, but the enzyme also fixes O2, which leads to the wasteful photorespiratory pathway. If we better understand the structure-function relationship of the enzyme, we might be able to engineer improvements. When the crystal structure of Chlamydomonas Rubisco was solved, four new posttranslational modifications were observed which are not present in other species. The modifications were 4-hydroxylation of the conserved Pro-104 and 151 residues, and S-methylation of the variable Cys-256 and 369 residues, which are Phe-256 and Val-369 in land plants. Because the modifications were only observed in Chlamydomonas Rubisco, they might account for the differences in kinetic properties between the algal and plant enzymes.

    METHODS: Site-directed mutagenesis and chloroplast transformation have been used to test the essentiality of these modifications by replacing each of the residues with alanine (Ala). Biochemical analyses were done to determine the specificity factors and kinetic constants.

    RESULTS: Replacing the modified-residues in Chlamydomonas Rubisco affected the enzyme's catalytic activity. Substituting hydroxy-Pro-104 and methyl-Cys-256 with alanine influenced Rubisco catalysis.

    CONCLUSION: This is the first study on these posttranslationally-modified residues in Rubisco by genetic engineering. As these forms of modifications/regulation are not available in plants, the modified residues could be a means to modulate Rubisco activity.

    GENERAL SIGNIFICANCE: With a better understanding of Rubisco structure-function, we can define targets for improving the enzyme.

    Matched MeSH terms: Genetic Engineering/methods
  18. Metabolic engineering of Lactococcus lactis influence of the overproduction of lipase enzyme
    Raftari M, Ghafourian S, Bakar FA
    J Dairy Res, 2013 Nov;80(4):490-5.
    PMID: 24063299 DOI: 10.1017/S0022029913000435
    The dairy industry uses lipase extensively for hydrolysis of milk fat. Lipase is used in the modification of the fatty acid chain length, to enhance the flavours of various chesses. Therefore finding the unlimited source of lipase is a concern of dairy industry. Due to the importance of lipase, this study was an attempt to express the lipase from Burkholderia cepacia in Lactococcus lactis. To achieve this, a gene associated with lipase transport was amplified and subcloned in inducible pNZ8148 vector, and subsequently transformed into Lc. lactis NZ9000. The enzyme assay as well as SDS-PAGE and western blotting were carried out to analysis the recombinant lipase expression. Nucleotide sequencing of the DNA insert from the clone revealed that the lipase activity corresponded to an open reading frame consisting of 1092 bp coding for a 37·5-kDa size protein. Blue colour colonies on nile blue sulphate agar and sharp band on 37·5-kD size on SDS-PAGE and western blotting results confirm the successful expression of lipase by Lc. lactis. The protein assay also showed high expression, approximately 152·2 μg/ml.h, of lipase by recombinant Lc. lactis. The results indicate that Lc. lactis has high potential to overproduce the recombinant lipase which can be used commercially for industrially purposes.
    Matched MeSH terms: Genetic Engineering
  19. Advances to improve the eating and cooking qualities of rice by marker-assisted breeding
    Phing Lau WC, Latif MA, Y Rafii M, Ismail MR, Puteh A
    Crit Rev Biotechnol, 2016;36(1):87-98.
    PMID: 24937109 DOI: 10.3109/07388551.2014.923987
    The eating and cooking qualities of rice are heavily emphasized in breeding programs because they determine market values and they are the appealing attributes sought by consumers. Conventional breeding has developed traditional varieties with improved eating and cooking qualities. Recently, intensive genetic studies have pinpointed the genes that control eating and cooking quality traits. Advances in genetic studies have developed molecular techniques, thereby allowing marker-assisted breeding (MAB) for improved eating and cooking qualities in rice. MAB has gained the attention of rice breeders for the advantages it can offer that conventional breeding cannot. There have been successful cases of using MAB to improve the eating and cooking qualities in rice over the years. Nevertheless, MAB should be applied cautiously given the intensive effort needed for genotyping. Perspectives from conventional breeding to marker-assisted breeding will be discussed in this review for the advancement of the eating and cooking qualities of fragrance, amylose content (AC), gel consistency (GC) and gelatinization temperature (GT) in rice. These four parameters are associated with eating and cooking qualities in rice. The genetic basis of these four parameters is also included in this review. MAB is another approach to rice variety improvement and development in addition to being an alternative to genetic engineering. The MAB approach shortens the varietal development time, and is therefore able to deliver improved rice varieties to farmers within a shorter period of time.
    Matched MeSH terms: Genetic Engineering
  20. Short homologies efficiently generate detectable homologous recombination events
    Osahor AN, Tan CY, Sim EU, Lee CW, Narayanan K
    Anal Biochem, 2014 Oct 1;462:26-8.
    PMID: 24929088 DOI: 10.1016/j.ab.2014.05.030
    When recombineering bacterial artificial chromosomes (BACs), it is common practice to design the ends of the donor molecule with 50 bp of homology specifying its insertion site. We demonstrate that desired recombinants can be produced using intermolecular homologies as short as 15 bp. Although the use of shorter donor end regions decreases total recombinants by several fold, the frequency of recombinants with correctly inserted donor molecules was high enough for easy detection by simple polymerase chain reaction (PCR) screening. This observation may have important implications for the design of oligonucleotides for recombineering, including significant cost savings, especially for high-throughput projects that use large quantities of primers.
    Matched MeSH terms: Genetic Engineering/methods*
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