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Construction of Metagenomic DNA Libraries and Enrichment Strategies

  • Farah Fadwa Benbelgacem
  • Hamzah Mohd. SallehEmail author
  • Ibrahim Ali Noorbatcha
Chapter

Abstract

Microorganisms are the main source of biocatalysts and with metagenomics approach it is now possible to overcome the major obstacle of microbiology which lies on the inability of the vast majority of microbes to grow under laboratory conditions. This chapter introduces and guides readers to a molecular biology approach that is useful for bioprospecting of novel, efficient and specific enzymes without escaping any unculturable or difficult to culture microorgaims. The first and necessary step of functional metagenomics is the metagenomic DNA library construction. It consists of metagenome extraction from the desired habitat, shearing of the DNA into sequences of desired size, end-repairing and cloning of these DNA sequences into a large-insert and high-copy number vector such as pCC1FOS with EPI300T1RE. coli as surrogate host, to the final step of storing the clones in glycerol stock for long term storage. Enrichment pre-DNA extraction step may be adopted to increase the likelihood that metagenomic DNA libraries hold the genes for the desired enzymatic activities.

Keywords

Metagenomic library Enrichment strategies Function-based metagenomic End-repair High-throughput screening 

Abbreviations and Chemical Formula

ATP

adenosine triphosphate

BACs:

bacterial artificial chromosomes

CMC

carboxyl methyl cellulose

DNA

deoxyribonucleic acid

dNTP

deoxyribonucleoside triphosphate

EDTA

ethylenediaminetetraacetic acid

EtBr

ethidium bromide

KCl

potassium chloride

K2HPO4

potassium hydrogen phosphate

LMP agarose

low melting point agarose

MgCl2.6H2O

magnesium chloride hexahydrate

MgSO4.7H2O

magnesium sulfate heptahydrate

NaCl

sodium chloride

Na2CO3

sodium carbonate

OD

optical density

PCR

polymerase chain reaction

POME

palm oil mill effluent

RNase

ribonuclease

TAE

tris-acetate-EDTA

TE

tris-EDTA

UV

ultraviolet

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Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • Farah Fadwa Benbelgacem
    • 1
  • Hamzah Mohd. Salleh
    • 1
    • 2
    Email author
  • Ibrahim Ali Noorbatcha
    • 1
  1. 1.Bioprocess and Molecular Engineering Research Unit, Kulliyyah of EngineeringInternational Islamic University MalaysiaKuala LumpurMalaysia
  2. 2.International Institute for Halal Research and TrainingInternational Islamic University MalaysiaKuala LumpurMalaysia

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