Advancements in Life Sciences (E-Journal, University of the Punjab)
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Cloning and expression of hepatitis B surface gene in E. coli
Get PDFBackground: Hepatitis B virus (HBV) is among the smallest DNA viruses resulting in ~800,000 deaths each year. Pakistan is considered a country affected by HBV. In Pakistan, the most dominant genotype is D. HBV is an enveloped virus of 3.2 kb. The study's goal was to express hepatitis B surface antigen in a bacterial host to produce a recombinant protein.Method: Blood samples were collected in EDTA coated vacutainer from patients after their consent. DNA was extracted from serum through the phenol-chloroform method; Hepatitis B surface gene was cloned in TA cloning vector, subclone in pET 28a expression vector. An expression vector containing the Surface gene was then transformed into a competent bacterial host BL21 and inducted with IPTG at 0.1-0.2mM concentration for expression. The expressed proteins (soluble and pellet form) were analyzed on SDS PAGE.Results: Hepatitis B Surface gene of 681bp after PCR were detected under UV light then successfully cloned and subcloned in pET 28 expression vector. The restricted fragment indicating the gene of interest was 681bp when analyzed on 1.2% Agarose gel under UV light. The required protein of 25kDa was obtained in soluble form when detected on 12% SDS PAGE after staining with Coomassie Blue dye.Conclusion: Hepatitis B surface gene was successfully expressed in both insoluble and pellet forms using E.coli. The expression of surface protein needs to maximize through optimizing conditions to be used as potent candidate for vaccine production to prevent hepatitis B infection.Keywords: Hepatitis B virus; Surface gene; Cloning vector; pET expression vector
CRISPR/Cas9 application in tomato breeding improvement: a review
Get PDFTomato (Solanum lycopersicum) is an essential plant because of its social and economic importance. Therefore, research have been focusing on improving tomato production. The introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (CRISPR/Cas9) system provides unique opportunities to better understand the gene functions and to rapidly generate new tomato cultivars harboring desired traits such as disease resistance, better harvest quality and abiotic tolerance. This review aims to provide latest information about the application of CRISPR/Cas9 system on tomato breeding.Keywords: CRISPR; Tomato breeding; Applied researc
Nitrogen removal efficiency of some bacterial strains isolated from seawater in Thua Thien Hue, Vietnam
Get PDFBackground: Nitrifying bacteria in aquaculture environments are capable of removing toxic nitrogen compounds such as ammonium and nitrite. Using these indigenous microbial resources can improve shrimp production.Methods: Screening method was used to isolate aerobic strains of nitrifying bacteria. Species identification for these isolates was done by biomolecular method based on 16S rDNA gene sequence. Ammonium, nitrite and nitrate concentrations from the culture were determined by spectrophotometry at the appropriate wavelength. Temperature, pH, dissolved oxygen and salinity were measured by specialized equipment. Formation and development of flocs during shrimp culture were determined based on their volume and weight. A trial of shrimp nursery was carried out on a small scale with 0.5 m3 tanks containing diluted seawater to 16-18‰ salinity at a density of 400 individual/m3 for 24 days on April 2019. Results: This study isolated two strains of Pseudomonas (BF01 and BF03) and one strain of Cupriavidus oxalaticus BF02 from seawater in Thua Thien Hue province, Vietnam. These bacterial isolates have shown ability to remove nitrogen compounds such as ammonium, nitrite and nitrate in culture medium. Formation and development of flocs were found in trials of shrimp nursery with diluted seawater containing the isolates. Some water quality parameters (temperature, pH, dissolved oxygen, salinity, ammonium and nitrite) were kept at a safe level and juvenile shrimp grown normally during culture.Conclusion: The observations on the water quality and basic growth parameters of juvenile shrimp in the two treatments, diluted seawater and diluted seawater with commercial microbial products, showed that there were no significant differences between them with p = 0.05. This proves that three isolates have played an important role in shrimp nursery. Keywords: Cupriavidus oxalaticus; Floc; Litopenaeus vanamei; Nitrifying-denitrifying bacteria; Pseudomonas sp.
Phosphorus Fertilizer Response to Onion (Allium cepa L.) Yield in Punjab, Pakistan
Get PDFBackground: Onion (Allium cepa L.) is one of the most essential plants in food with high nutritional value. However, application of right dose of phosphorous (P) is one of the constraints to the profitable onion yields in soils deficient in P.Methods: A systematic study to confirm the best dose of P was conducted for six years in the P deficient soil in farmers’ fields. Based on the findings obtained from 2008-09 to 2010-11, the research was undertaken to determine the effect of different phosphorus levels on the yield of onion in the Randomized Complete Block Design (RCBD) with a total of 114 replicates in 2011-12 to 2012-13. Four treatments (160, 210, 260 and 310 kg P2O5 ha-1) were tested with N and K at 100 kg ha-1.Results: From the results of this investigation, the variance analysis showed the substantial P impact. The maximum marketable bulb yield (19.03 t ha-1) was obtained from the fertilizer combination NPK @ 100-310-100 kg ha-1 and was shown to be statistically higher than all other treatments.Conclusion: Nonetheless, the nutshell of the overall economic study is that poor farmers (Land holders >12 acres) may have options to select the NPK fertilizer combination @ 100:210:100 kg ha-1 and the average farmer may have options to select the NPK fertilizer combination @ 100:260:100 kg ha-1. But rich farmers (Land holders >25 acres) who can spend more money on fertilizers and are interested in the higher gross margin should follow the combination of NPK fertilizers @ 100:310:100 kg ha-1 to profitably increase their gross margin and maintain soil fertility for onion cultivation in Punjab, Pakistan. Keywords: Onion; NPK; Plant nutrition; Phosphorus; Pungency
Prevalence of Colibacillosis in Young Broiler Chicks and Antibiogram of Escherichia coli in Different Areas of Hazara Region
Get PDFBackground: Poultry sector is a vital zone of Pakistan economy and is a feasible source of animal derivative protein. In term of mortality and morbidity the development of this segment is significantly affected by a lot of problems. Hazara region is well known for poultry in Pakistan. Colibacillosis, caused by Escherichia coli is vital disease among poultry of all ages resulting in huge economic losses. The aim of this study was to evaluate the prevalence of colibacillosis in young broiler chickens and antibiogram of E. coli in different areas of Hazara region.Methods: The current study was conducted to evaluate the prevalence of colibacillosis in young broiler chickens and antibiogram of E. coli. This study was done during February 2019 to June 2019; a total of 200 (n=200) liver samples of freshly dead young broiler chicks were randomly collected from the postmortem section of Veterinary research and disease investigation center Abbottabad (VRDICA). The identification of E. coli was confirmed by gram staining and biochemical tests. Susceptibility pattern to 13 antibiotics was also checked.Results: Total of 200 (n=200) samples were brought to the VRDICA for the diagnosis of disease and antibiotic susceptibility testing. Out of these 130 were positive (Overall prevalence =65%).Area wise prevalence varied from area to area and highest prevalence was recorded in Abbottabad (81.11%) followed by Havelian (75%), Manghal (72.22%), Mansehra (60%), Qalandarabad (50%) and Haripur (28.57%). About age group, highest mortality was found in 11-15 days old chicks (93%) as compared to 6-10days (83.33%) and 1-5 days old chicks (21.42%). Antibiogram showed highest sensitivity to Gentamicin (100%) afterward Colistin Sulphate (92.30%). However, lincomycin (92%) and streptomycin (96%) display highest resistance.Conclusion: In this study, the highest prevalence of infection was observed in young chicks that play a crucial role in their mortality. E. coli had showed resistance to commonly recommended antibiotic so care should be taken while prescribing the drugs.Keywords: Young broiler chicks; E. coli; Colibacillosis; Prevalence; Antibiogram
Probiotic potential of encapsulated Lactobacillus species in yogurt formation indigenously isolated from dairy source
Get PDFBackground: Exploration of beneficial bacteria as probiotics inputs are drawing interest in dairy industry but their long-term survival and viability is an important consideration.Methods: The current work focused on the exploration of probiotic potential of indigenously isolated Lactobacillus strains from dairy products and their encapsulation and utilization in yogurt formation.Results: These Lactobacillus strains were identified as Lactobacillus fermentum MGA23-1 and Lactobacillus fermentum LMEM19 and found resistant to inhibitory substances like phenol (0.2%), bile salts (0.3%), pancreatin (0.5%) and pepsin (0.3%). The highest antibacterial activity was observed by Lactobacillus fermentum MGA23-1 against Pseudomonas aeruginosa (13mm). Encapsulation experiment showed that the number of bacterial CFU/g increased significantly (p < 0.05) in beads during storage up to 7 days. Chemical characterization of microcapsules was assessed using FTIR and showed characteristics wavelength major at 1541 – 1716 cm-1 and 3336 cm-1. Yogurt was prepared using a single probiotic strain, in a consortium of Lactobacillus fermentum MGA23-1 and Lactobacillus fermentum LMEM19 and in the form of beads. Best results were observed in the case of microencapsulation.Conclusion: It was concluded that both strains had the potential to be used as a probiotic in the dairy industry.Keywords: Probiotic; Tolerance to inhibitory substances; Antibacterial activity; Adhesion ability; Safety; Microencapsulation; FTIR
Narrative Review: Use of Competent Stimulating Peptide in Gene Transfer Via Suicide Plasmid in Streptococcus pneumoniae
Get PDFNatural competency for genetic transformation of Streptococcus pneumoniae causes the emergence of novel or non-vaccine preventable pneumococcal serotypes. This phenomenon has become a global concern as it can spread quickly in the population through inhalation and close contact. The colonisation of S. pneumoniae at the upper respiratory tract can either become commensal or pathogenic. Once the bacterium invades into the body system, it will secrete its toxin and virulence protein to facilitate the invasion. Besides, S. pneumoniae can undergo natural biological transformation via uptake of exogenous DNA by horizontal gene transfer for integration and recombination of the genome. S. pneumoniae natural transformation is aided by competence-stimulating peptide (CSP) that induces the competence of bacteria. Natural transformation cascade of S. pneumoniae via CSP is triggered in the presence of conserved 17-amino acids peptide which is regulated and encoded by comC, comD and comE operon, where comC is responsible in secreting precursor CSP. Nowadays, researchers transforming S. pneumoniae by inserting the mutated S. pneumoniae gene through a vector, suicide plasmid. Suicide plasmids such as pID701, pAUL-A and pVA891 can be transferred but cannot replicate in the bacteria. Homologous recombination process occurs once the mutated gene of suicide plasmid is integrated with wild-type S. pneumoniae. Previous studies had used the transformation of suicide plasmid into S. pneumoniae as it can integrate with host DNA at specific insert for gene transfer. But there is no evidence on the role of CSP in horizontal/gene replacement via suicide plasmid in Streptococcus pneumoniae. This narrative review's scope as per defined purpose statement is to relate and recommend the use of competent stimulating peptide in efficient horizontal gene transfer via suicide plasmids in Streptococcus pneumoniae.Keywords: Streptococcus pneumoniae transformation; CSP-based transformation; Gene transfer via suicide plasmid; Genetic exchange; Competence-stimulating peptid
The Effect of Drought Stress on the Superoxide Dismutase and Chlorophyll Content in Durum Wheat Genotypes
Get PDFBackground: Drought stress is one of the most limiting factors of plant production around the world. So, finding a way for increasing genotypes resistance is so important. Free radicles and other dynamic subordinates of oxygen inactivate chemicals and significant plant cell parts. Superoxide dismutases (SODs) have been distinguished as essential parts in a creature's guard system.Methods: This examination was carried out to examine the SOD movement in 8 durum wheat genotypes from Iran and Azerbaijan under two different conditions in 2015-2016 cropping year. The impact of dry season weight on SOD, chlorophyll content list (CCI), and chlorophyll debasement were examined. Critical contrasts among genotypes and the genotype × climate collaboration among SOD and CCI content were distinguished.Results: The mean examination indicated that the substance of SOD and CCI diminished in susceptible genotypes, while tolerant genotypes SOD and CCI stayed unaltered or increased. For measuring drought tolerance, the stress tolerance index (STI) used. The correlation between STI for Chlorophyll and Chlorophyll CI in drought was significant at 0.01 levels. The pressure resilience list (STI) for SOD and CCI characterized safe and defenseless genotypes into unmistakable gatherings.Conclusion: Hence, these 2 characters can be utilized as a Selection index for screening dry spell safe plant materials.Keywords: Durum wheat, Drought stress, SOD, ST
Role of commercially available SARS-CoV-2 detection kits in pandemic of COVID-19 on the basis of N and E gene detection
Get PDFCoronavirus has blowout worldwide from the time when its revelation in Hubei Province, China in December 2019 introducing a genuine general wellbeing emergency. The capacity to recognize an irresistible specialist in a broad pestilence is vital to the achievement of isolate endeavors notwithstanding the delicate and precise screening of expected instances of disease from patients in a clinical setting. Structural proteins the basic key role-playing in SARS-CoV2 identification include a spike, envelope membrane, nucleocapsid, and helper proteins. N-protein ties to the infection single positive-strand RNA that permits the infection to assume control over human cells and transform them into infection industrial facilities inside the capsid and E-protein shows a significant part in infection gathering, film permeability of the host cell, and infection has cell correspondence. Nucleic-Acid base testing presently offers the most touchy and early discovery of COVID-19. Notwithstanding, analytic advancements have explicit impediments and announced a few false negative and false positive cases, particularly during the beginning phases of contamination. Presently, more refined diagnostics are being created to improve the COVID-19 determination. This article presents an outline of diagnostic approaches to address a few inquiries and issues identified with the constraints of flow innovations and future innovative work difficulties to empower ideal, fast, minimal effort, and precise analysis of arising irresistible illnesses We depict purpose of-care diagnostics that are not too far off and urge scholastics to propel their advancements past origination. Creating fitting and-play diagnostics to deal with the SARS-CoV-2 flare-up would be valuable in forestalling forthcoming pandemics.Keywords: Role of commercially available kits; SARS-CoV2; Pandemic of Covid-19; N gene; E gen
Characterization and Quantification of Betasatellites Research from Google Scholar Using a Bibliometric Approach
Get PDFBackground: Begomoviruses belongs to the family Geminiviridae. These viruses are associated with various satellite DNA molecules (alpha satellites, delta satellites, and betasatellites).Methods: The standardized search strategy was applied to get the research output regarding betasatellites worldwide for bibliometric analysis. Google scholar database was used to collect the data regarding betasatellites research from past 1997 to June 2018.Results: A total of 213 documents regarding the studied subject were identified, out of which the maximum number of documents 153 (71.83%) were found to have been published in the form of original research articles. The common language of publications 208 (97.65%) was English. The results declare that the number of publications shows an increase from 1997 to 2014, with the maximum number of publications in 2014. The top 10 productive countries based on the maximum number of publications were India 83 (38.96%), Pakistan 66 (30.98%), China 26 (12.20%) followed by some other countries. USA and Oman were the top collaborative countries with Pakistan respectively.Conclusion: For all the analyzed documents the total number of citations was 3564 with an average of 16.73 citations per document. The journal “Virus Genes” was the most prolific journal based on a maximum number of publications on the studied subject. Robert William Briddon and Shahid Mansoor were the most productive scientists, and the National Institute for Biotechnology and Genetic Engineering (NIBGE) was the most productive institute regarding the studied subject. This study shows that India, Pakistan, the USA, and China play an important role in the area of beta satellite research.Keywords: Betasatellites; Bibliometric Analysis; Begomoviruses