bioprospecting environmental metagenomes
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Bioprospecting Environmental metagenomes - BTech Honours report
1.0 Abstract
Microorganisms dominate the biosphere, yet current methods of culture reveal less than1% of the microbial diversity. This hidden diversity contains a vast array of novelenymes and antimicrobials. !e" enymes could potentially be useful to manyindustrial applications, and ne" antimicrobial drugs are becoming increasinglyimportant.
Techni#ues such as those detailed in this proposal, present one "ay of accessing orbioprospecting this hidden unculturable diversity. The essence behind these methods isto e$tract the !& from a given environment 'activated sludge "as used here(. Theninsert a fragment of this !& into an e$pression plasmid, creating a )metagenomic)
library. Then to functionally assay the e$pressed !& for a multitude of activities'lipase, cellulase, protease, hemicellulase, and antimicrobial activities "ere planned(
*nsufficient time ho"ever, did not allo" completion of this pro+ect. roblems "ereencountered "ith plasmid amplification and obtaining good #uality high molecular"eight !& from the activated sludge samples. olutions and suggestions for these
problems are discussed.
2.0 Acknowledgements
* "ould lie to than my supervisors avid aul and usan Turner for their support andguidance throughout the course of this pro+ect.
Than you to /herida Trott, 0im-y /hhour, hingo Miyauchi, and &ndre" 2errie"ho "elcomed me into the lab and sho"ed me the ropes, and ans"ered my 3o "hereis the 43 and 3Ho" do you 43 #uestions.
3.0 Contents
1.0Abstract
2.0Acknowledgements
3.0Contents
4.0Introduction
4.1Bioprospecting and the Metagenome
4.2i!ersit" and the #$nculturables#
4.3%re!ious &ork
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4.4Aims
4.'Methodolog"
4.'.1Multiple( Cloning
4.'.1.1)ample Collection
4.'.1.2*A +(traction
4.'.1.3Insert *A %reparation
4.'.1.4,he p+, -ector )"stem
4.'.1.'igation
4.'.1./,ransormation
4.'.2+(pression )creening
4.'.2.1"drol"tic +n"me Assa"s
4.'.2.2Antimicrobial Assa"s
4.'.2.3Combinational Assa"s
4.'.3Insert Characterisation
4.'.3.1%lasmid eco!er"
4.'.3.2)euencing o Insert
4./)igniicance o 5utcomes
4./.1ipases
4./.2emicellulases
4./.3Antimicrobials
'.0esults
'.1%lasmid Ampliication
'.2*A +(traction
'.3*A igestion
'.4igation and ,ransormation
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/.0iscussion
/.1%lasmid Ampliication iiculties
/.2igh 6ualit" *A +(traction iiculties
/.2.1Bead7Beater +(traction
/.2.2igestion o Metagenomic *A
/.2.38)hort %rotocols8 Bacterial9 :enomic *A +(traction
/.2.4igh Molecular &eight *A +(traction rom )oil ;%orteous et al.0eerences
?.0Appendices
?.1,erms and Appendices
?.2Methods
?.2.1*A +(traction Methods
?.2.1.1Bead7beater *A +(traction Method
?.2.1.28)hort %rotocols8 Bacterial :enomic *A +(traction
?.2.1.3igh Molecular &eight *A +(traction rom )oil ;%orteous et al147/.
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:riithsF G. 2001. Methods. Macuarie $ni!ersit"F )"dne". ;%ersonal
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genes conerring utiliation o 47 h"dro("but"rate'pplied and (nironmental
Microbiology. /'3?017=.
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di!ersit" o uncultured microorganisms'pplied (nironmental Microbiology.
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ondonF M. .F ). H. aelF . M. :oodmanF and H. andelsman 1???. ,oward
unctional genomics in bacteria anal"sis o gene e(pression in +scherichia coli
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the National 'cademy of Sciences !S'. ?//4'17'.
)chutF %. 1??/. umic acids.'griculture and 'gri-ood anada. J&eb %ageKhttpEEsis.agr.gc.caEcansisEglossar"EhumicLacid.html JAccessed 2/ Hul" 2001K
,eatherF . M.F and %. H. &ood 1?>2. $se o Congo red7pol"saccharide
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bo!ine rumen.'pplied and (nironmental Microbiology. 43===7>0.
,ebbeF C. C.F and &. -ahen 1??3. Intererence o humic acids and *A e(tracted
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bacteria and a "east'pplied and (nironmental Microbiology. '?2/'=72//'.
,urnerF ). 2001. %)C7B ;%hosphateF ))F Chloroorm7Bead Beater< method or*A e(traction rom en!ironmental samples. &he !niersity of 'uckland.
;%ersonal Communication
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