Faiza Saleem

In Vivo Crystallization of Three-Domain Cry Toxins

Bacillus thuringiensis (Bt) is the most successful, environmentally-friendly, and intensively studied microbial insecticide. The major characteristic of Bt is the production of proteinaceous crystals containing toxins with specific activity against many pests including dipteran, lepidopteran, and coleopteran insects, as well as nematodes, protozoa, flukes, and mites. These crystals allow large quantities of the protein toxins to remain stable in the environment until ingested by a susceptible host. It has been previously established that 135 kDa Cry proteins have a crystallization domain at their C-terminal end. In the absence of this domain, Cry proteins often need helper proteins or other factors for crystallization. In this review, we classify the Cry proteins based on their requirements for crystallization.

Molecular characterisation of Bacillus chitinase for bioconversion of chitin waste

In this work chitin was extracted chemically from shrimp shells. Seventeen Bacillus isolates were screened for chitinolytic activity. The chitinolytic strains of Bt. were screened at different temperatures and pHs for their hydrolytic potentials. By using a pair of specific primers, endochitinase gene was amplified from SBS Bt-5 strain through PCR, and then cloned into pTZ57 TA cloning vector and transferred in Escherichia coli DH5α strain. The sequenced gene (GenBank Accession No: HE995800) consists of 2031 nucleotides capable of encoding 676 residues. The protein consisted of three functional domains with a calculated molecular mass of 74.53 kDa and a pI value of 5.83. The amino acid sequence of chi gene showed 99% similarity to the genes of Bt MR11 endochitinase, Bt serovar kurstaki chitinase (kchi), Bt strain MR21 endochitinase and Bacillus cereus B4264.

Mass cultivation of various algal species and their evaluation as a potential candidate for lipid production

Microalgae have been proposed as a promising source for biodiesel production. Focusing on algal strains for biodiesel production, efforts should be made to search new strains. Experiments were carried out to investigate the effects of growth parameters (nutrients, pH, light, aeration and temperature) and the oil percentage of eight algal strains (Chlorella sp., Cladophora sp., Hydrodictylium sp., Oedogonium sp., Oscillatoria sp., Spirogyra sp., Stigeocolonium sp., Ulothrix sp.). Results show that 6.5–7.5 is the optimum pH for the growth of all algal species. Temperature showed a greater variation (25°40°C). Ulothrix sp. gave more biomass productivity and is the most suitable strain for biodiesel production due to higher oil percentage (62%). Least biomass production was observed for Stigeocolonium sp. and least oil content was obtained from Hydrodictylium sp. It was observed that among these eight algal strains for biodiesel production, Ulothrix and Chlorella are the most promising algae species.

The First Cry2Ac-Type Protein Toxic to Helicoverpa armigera: Cloning and Overexpression of Cry2ac7 Gene from SBS-BT1 Strain of Bacillus thuringiensis

The Cry (crystal) proteins from Bacillus thuringiensis are known to have toxicity against a variety of insects and have been exploited to control insect pests through transgenic plants and biopesticides. B. thuringiensis SBS BT-1 carrying the cry2 genes was isolated from soil samples in Pakistan. The 2-kb full length cry2Ac gene was cloned, sequenced, and submitted to the EMBL DNA database (Accession No. AM292031). For expression analysis, Escherichia coli DH5α was transformed with the fragment sub-cloned in pET22b expression vector using NdeI and HindIII restriction sites, and later confirmed by restriction endonuclease analysis. To assess the toxicity of Cry2Ac7 protein against lepidopteran and dipteran insects, BL21 (codon plus) strain of E. coli was further transformed with the recombinant plasmid. The 65-kDa protein was expressed in the form of inclusion bodies up to 180 OD units per liter of the medium. Inclusions were washed with a buffer containing 1.5% Triton-X 100 and >90% pure Cry2Ac7 was obtained. The inclusion bodies were dissolved in 50 mM K2CO3 (pH 11.5), dialyzed, and freeze-dried. This freeze-dried protein as well as inclusion bodies were used in bioassays against larvae of Helicoverpa armigera and Musca domestica. The freeze-dried protein was toxic to H. armigera larvae with an LC50 value of 131 ng/mL. However, Cry2Ac7 produced in E. coli did not show any mortality to M. domestica larvae. This is the first report of Cry2Ac protein toxic to H. armigera.

Multiple upstream start codons (AUG) in 5′ untranslated region enhance translation efficiency of cry2Ac11 without helper protein: ADALAT et al.

Cry2Ac11, a 65 kDa insecticidal protein produced by Bacillus thuringiensis, shows toxicity against dipteran and lepidopteran larvae. It is encoded by cry2Ac11 gene ( orf3), which is part of an operon comprising orf1, orf2, and orf3. Orf2, a helper protein, helps in proper folding and prevents aberrant aggregation of newly produced molecules. In this study, we have elucidated the effect of different mutations in translation initiation region (TIR), particularly the ribosomal binding site and the start codon (RBS‐ATG) on cry2Ac11 gene expression without helper protein. All recombinant constructs were expressed in acrystalliferous B. thuringiensis subsp israelensis 4Q7 under the control of strong chimeric promoter cyt1AP/STAB. Of all the mutants, mut/RBS2, with two consecutive AUGs after the spacer region in TIR, exhibited 89‐ and 2246‐fold higher transcript levels compared with 4Q7‐operSalI/RBS ( cry2Ac11 operon) and 4Q7‐w‐RBS ( cry2Ac11 gene), respectively. The analysis of mut/RBS2 messenger RNA (mRNA) structure in the RBS‐AUG region showed the presence of RBS in the single‐stranded part of the moderately stable hairpin loop. The high expression efficiency of Cry2Ac11 mutant without helper protein is a cumulative and cooperative result of chimeric promoter cyt1AP/STAB‐SD with the optimal context of RBS‐AUG region provided by multiple AUGs and stabilizer sequence at 3′ ends.

In vitro evaluation of mutagenicity and genotoxicity of sitagliptin alone and in combination with artificial sweeteners

Purpose: To determine the in vitro genotoxicity and mutagenicity of sitagliptin alone and in combination with three commonly used artificial sweeteners (saccharin, aspartame and acesulfame-k). Methods: The in vitro genotoxicity and mutagenicity of Sitagliptin alone and in combination with three popular artificial sweeteners (saccharin, aspartame and acesulfame-k) were evaluated by Comet and Ames assays, respectively. Results: Sitagliptin demonstrated mutagenic potential only to TA 98 with S9 mix at a concentration of 3040 μg/plate. The mutagenicity of sitagliptin was enhanced when tested in combination with the artificial sweeteners. Furthermore, sitagliptin also caused pronounced DNA fragmentation at higher doses compared with negative control. Conclusion: At higher doses, sitagliptin showed both mutagenicity and genotoxicity. Thus, long-term use of artificial sweeteners with sitagliptin may lead to increase in both mutagenicity and genotoxicity. Keywords: Sitagliptin, Artificial sweeteners, Comet assay, DNA damage, Ames assay, Genotoxicity, Mutagenicity