Boundaries in metagenomic screenings using lacZ alpha-based vectors

Autor(es)

Alves, Luana de Fatima ^{[1, 2]} ; Borelli, Tiago Cabral ^[2] ; Westmann, Caua Antunes ^[3] ; Silva-Rocha, Rafael ^[3] ; Guazzaroni, Maria-Eugenia ^[2]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Fac Med Ribeirao Preto, Dept Bioquim & Imunol, Ribeirao Preto, SP - Brazil

^[2] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Biol, Av Bandeirantes 3-900, BR-14049901 Ribeirao Preto, SP - Brazil

^[3] Univ Sao Paulo, Fac Med Ribeirao Preto, Dept Biol Celular & Mol, Ribeirao Preto, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: GENETICS AND MOLECULAR BIOLOGY; v. 43, n. 1 2020.
• Citações Web of Science: 0

Resumo

Metagenomics approaches have been of high relevance for providing enzymes used in diverse industrial applications. In the current study, we have focused on the prospection of protease and glycosyl hydrolase activities from a soil sample by using the lacZ alpha -based plasmid pSEVA232. For this, we used a functional screen based on skimmed milk agar and a pH indicator dye for detection of both enzymes, as previously reported in literature. Although we effectively identified positive clones in the screenings, subsequent experiments revealed that this phenotype was not because of the hydrolytic activity encoded in the metagenomic fragments, but rather due to the insertion of small metagenomic DNA fragments in frame within the coding region of the lacZ gene present in the original vector. Analyses of the thermodynamic stability of mRNA secondary structures indicated that recovering of positive clones was probably due to higher expression levels of the chimeric lacZ alpha-genes in respect to the original from empty vector. We concluded that this method has a higher tendency for recovery false positive clones, when used in combination with a lacZ alpha-based vector. As these vectors are massively used in functional metagenomic screenings, we highlight the importance of reporting boundaries in established metagenomic screenings methodologies. (AU)

• Processo FAPESP: 15/04309-1 - Novas abordagens para melhorar a prospecção funcional de biocatalizadores em bibliotecas metagenômicas
• Beneficiário: María Eugenia Guazzaroni
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores
• Processo FAPESP: 16/05472-6 - Engenharia e prospecção de novos elementos regulatórios em bactéria através de abordagens de biologia sintética
• Beneficiário: Cauã Antunes Westmann
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 17/20818-9 - Caracterização de novos elementos regulatórios metagenômicos
• Beneficiário: Tiago Cabral Borelli
• Modalidade de apoio: Bolsas no Brasil - Iniciação Científica
• Processo FAPESP: 16/06323-4 - Novas ferramentas para abordagens metagenômicas na prospecção de celulases
• Beneficiário: Luana de Fátima Alves
• Modalidade de apoio: Bolsas no Brasil - Doutorado