Quantifying the antimicrobial activity of CRISPR-Cas9-accA modified ΔB. subtilis mutants against V. harveyi and E. Coli

Probiotics are increasingly popular, currently. Probiotics have been described with the ability to treat many disorders of the gastrointestinal tract (GIT) such as irritable bowel syndrome (IBS)and Crohns disease. Types of probiotics include bacterial strains from Lactobacillus and Bifidobacterium. Probiotics can restore balance to gut microbiota by outcompeting pathogenic bacteria for nutrients and secrete antimicrobials to eliminate these bacterial pathogens. However, the viability of most advertised probiotics lose their potency due to being freeze dried into powders during storage or for consuming. Many probiotics become ineffective and produce lower CFUs while traversing through the gastric acids of the digestive system. For these reasons, this study sought to enhance the antimicrobial response of a highly potent probiotic known as Bacillus subtilis. B. subtilis has been used to treat many disorders of the gut and secrete many antimicrobials lethal for pathogenic microbes. B. subtilis was genetically modified to express CRISPR-Cas9 nuclease deletion of the accA gene B.subtilis mutants, which inhibits expression of an essential accA gene a part of the fatty acid synthesis (FAS) metabolic pathway. The CRISPR-Cas9-accA B.subtilis mutants were co-cultured with V. harveyi and E. Coli. Bacterial growth, biofilm formation, antimicrobial activity, and antibiotic resistance were quantified. It was found that B. subtilis mutants co-cultured with V. harveyi and E. Coli lessened bacterial growth, amplified biofilm with V. harveyi, reduced biofilm formation of E. Coli, the co-cultures with the mutants lacked antimicrobial activity, and increased the antibiotic resistance of V. harveyi and E. Coli. It can be concluded that there is an immense potential for using genetically engineered probiotic strains to enhance the antimicrobial activity of B. subtilis, which can amplify the reduction of pathogenic bacteria. However, the safety and frugality of using B. subtilis as a probiotic requires further consideration.

Cloning and Expression Analysis of Carboxyltransferase of Acetyl-CoA Carboxylase from Jatropha curcas

A full-length cDNA of the carboxyltransferase (accA) gene of acetyl-coenzym A (acetyl- CoA) carboxylase from Jatropha curcas was cloned and sequenced. The gene with an open reading frame (ORF) of 1149 bp encodes a polypeptide of 383 amino acids, with a molecular mass of 41.9 kDa. Utilizing fl uorogenic real-time polymerase chain reaction (RT-PCR), the expression levels of the accA gene in leaves and fruits at early, middle and late stages under pH 7.0/8.0 and light/darkness stress were investigated. The expression levels of the accA gene in leaves at early, middle and late stages increased signifi cantly under pH 8.0 stress compared to pH 7.0. Similarly, the expression levels in fruits showed a signifi cant increase under darkness condition compared to the control. Under light stress, the expression levels in the fruits at early, middle and late stages showed the largest fl uctuations compared to those of the control. These fi ndings suggested that the expression levels of the accA gene are closely related to the growth conditions and developmental stages in the leaves and fruits of Jatropha curcas.