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 EPI300T1R E. 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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Abbreviations
- 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
References
Carola S, Rolf D (2011) Metagenomic analyses: past and future trends. Appl Environ Microbiol 77(4):1153–1161
Cowan D, Meyer Q, Stafford W, Muyanga S, Cameron R, Wittwer P (2005) Metagenomic gene discovery: past, present and future. Trends Biotechnol 23(6):321–329
Handelsman J, Rondon MR, Brady SF, Clardy J, Goodman RM (1998) Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem Biol 5:R245–R249
Kauffmann IM, Schmid RD, Schmitt J (2004) DNA isolation from soil samples for cloning in different hosts. Appl Microbiol Biotechnol 64:665–670
Kennedy J, O’Leary ND, Kiran GS, Morrissey JP, O’Gara F, Selvin J, Dobson ADW (2011) Functional metagenomic strategies for the discovery of novel enzymes and biosurfactants with biotechnological applications from marine ecosystems. J Appl Microbiol 111:787–799
Praveen K, Yasuhisa A (2012) Strategies for discovery and improvement of enzyme function: state of the art and opportunities. Microb Biotechnol 5:18–33
Rees HC, Grant S, Jones B, Grant WD, Heaphy S (2003) Detecting cellulase and esterase enzyme activities encoded by novel genes present in environmental DNA libraries. Extremophiles 7:415–421
Sabree ZL, Rondon MR, Handelsman J (2009) Metagenomics. In: Schaechter M (ed) Encyclopedia of microbiology, 3rd edn. Elsevier, Amsterdam
Voget S, Leggewie C, Uesbeck A, Raasch C, Jaeger KE, Streit WR (2003) Prospecting for Novel Biocatalyst in a Soil Metagenome. Appl Environ Microbiol 7:6235–6242. Epicentre: http://www.epibio.com/
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Nature Singapore Pte Ltd.
About this chapter
Cite this chapter
Benbelgacem, F.F., Salleh, H.M., Noorbatcha, I.A. (2018). Construction of Metagenomic DNA Libraries and Enrichment Strategies. In: Amid, A., Sulaiman, S., Jimat, D., Azmin, N. (eds) Multifaceted Protocol in Biotechnology. Springer, Singapore. https://doi.org/10.1007/978-981-13-2257-0_3
Download citation
DOI: https://doi.org/10.1007/978-981-13-2257-0_3
Published:
Publisher Name: Springer, Singapore
Print ISBN: 978-981-13-2256-3
Online ISBN: 978-981-13-2257-0
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)