bioprospecting environmental metagenomes

5/28/2018 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/.

:ibbsF M. . 1??'. %.. ,hesis. emicellulases rom e(tremel" thermophilic

microorganisms as wood pulp bleaching aids. &he !niersity of 'uckland.


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rontier or natural products$ournal of %iological hemistry. '24'7?.

awkesF ). . H. 1??>. %.. ,hesis. Molecular )tudies o Adrenoleukod"stroph"

and +mer"7reiuss Muscular "stroph". &he !niersity of 'uckland.

enneF A.F . anielF . A. )chmitF and :. :ottschalk 1???. Construction o

en!ironmental *A libraries in +scherichia coli and screening or the presence o

genes conerring utiliation o 47 h"dro("but"rate'pplied and (nironmental

Microbiology. /'3?017=.

enneF A.F . A. )chmitF M. BomekeF :. :ottschalkF and . aniel 2000.

)creening o en!ironmental *A libraries or the presence o genes conerringlipol"tic acti!it" on +scherichia coli'pplied and (nironmental Microbiology.

//31137/.

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Harbor )abratoryF *ew ork.

MillerF . *.F H. +. Br"antF +. . MadsenF and &. C. :hiorse 1???. +!aluation and

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samples'pplied and (nironmental Microbiology. /'4=1'724.

MorrisF . . 1??=. %.. ,hesis. Isolation o ("lanase genes rom e(tremel"

thermophilic bacteria as potential krat pulp bleaching aids. &he !niersity of

'uckland.

%orteousF . A.F H. . ArmstrongF . H. )eidlerF and . ). &atrud 1??4. An eecti!e

method to e(tract *A rom en!ironmental samples or pol"merase chain

reaction ampliication and *A ingerprint anal"sis urrent Microbiology. 2?3017

=.

%rescottF . M.F H. %. arle"F and . A. Glein 1???. Microbiolog". *% Mc+ra-

HillF Boston.

ondonF M. .F %. . AugustF A. . BettermannF ). . Brad"F ,. . :rossmanF M.

. ilesF G. A. oiaconoF B. A. "nchF I. A. Mac*eilF C. MinorF C. . ,iongF M.

:ilmanF M. ). 5sburneF H. Clard"F H. andelsmanF and . M. :oodman 2000.

Cloning the soil metagenome a strateg" or accessing the genetic and unctional

di!ersit" o uncultured microorganisms'pplied (nironmental Microbiology.

//2'417=.

ondonF M. .F . M. :oodmanF and H. andelsman 1???. ,he +arth#s bount"

assessing and accessing soil microbial di!ersit" &rends in %iotechnolology. 1=4037

?.


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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

rom a bacterial artiicial chromosome librar" o Bacillus cereus#roceedings of

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

interactions in enumeration and characteriation o cellulol"tic bacteria rom the

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

directl" rom soil in detection and transormation o recombinant *A rom

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.

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bioprospecting environmental metagenomes

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