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Oil - degradative capacities
Sample chosen: U3
Objectives
Evaluation of major microbial groups
Background for applications
Adjusting protocols
2. 16S rRNA gene libraries
Oil issue:
1. Enrichment with crude oil, isolation
3. Metagenomes:“Archive” libraries, Expression libraries
Three pathways for the metabolic attack on n-alkanes are known:
Molecular genetics and pathway for alkane oxidation
ii) biterminal oxidation ( of several types of bacteria and fungi)
H3CRCH3 ->H3CRCH2OH -> HOCH2RCH2OH -> HOOCRCOOH
iii) subterminal oxidation (Nocardia spp.) RCH2CH3 -> RCH(OH)CH3 -> RC(O)CH3
i) monoterminal oxidation pathway (Pseudomonas spp.)
RCH3 -> RCH2OH -> RCHO -> RCOOH
Enrichment (Urania interphase)
Pure cultures
Dilutions, plating
Metagenomes
DNA prep
PCR, cloning
Re-amplification
RFLP-Clustering,sequencing sequence analysis
Oil enrichment "O": selected for sequencing1 2 3 4 5 6 7 8 9 10 11 12
A 1 2 3 4 5 6 7 8 9 10 11 12B 13 14 15 16 17 18 19 20 21 22 23 24C 25 26 27 28 29 30 31 32 33 34 35 36D 37 38 39 40 41 42 43 44 45 46 47 48E 49 50 51 52 53 54 55 56 57 58 59 60F 61 62 63 64 65 66 67 68 69 70 71 72G 73 74 75 76 77 78 79 80 81 82 83 84H 85 86 87 88 89 90 91 92 93 94 95 96
Incubation for six weeks at r.t.
0.1 substitution/site
Thiomicrospiragroup
Chromatiaceae
Legionellaceae
CardiobacteriaceaeMethylococcaceaeEnterobacteriaceaePasteurellaceae
Vibrionaceae
Aeromonasgroup
Colwelliagroup
Xanthomonas- Stenotrophomonas group
Microbulbifer
Neptunomonas
Marinomonas
Alcanivorax
Oleiphilus messinensis
Oceanospirillum
Marinobacter
Pseudomonas(sensu stricto)
Isolates from oil enrichment.
Ps. stutzeri-Ps. balearica groupMarinobacter hydrocarbonoclasticus-Mb.CABProteobacteria (Ruegeria group)
METAGENOMIC LIBRARIES
pBAC (e.g. pBeloBac11) large fragments 50-300 kbp
SuperCos1 - a „common“cosmid for cloning, Fragments to clone 40-50 kbp
“Archives”
based systems, e.g. LambdaZap
Expression libraries
pLAFR3 - a replicon in a variety of Gram-negatives(e.g. P. putida)fragments to clone - 20-30 kbp
Strategy to construct genomic library in cosmid vectors pLAFR3/SuperCos
Cosmid arms treated with phosphatese
Transduce, select for antibiotics resistans and score for white colonies on X-Gal
DNA size-fractionated,partially digested with Sau3A
Ligation
Package in vitro
Library of dozens of thousands arrayed clones with 20-50 kbp inserts
Cosmid library (contd.)
Arrayed clonesColony picking
Sequencing of selected clones
Subcloning/expres-sion of gene of interest
Probing
Enzymes Detection systemEsterases/proteases
Phosphatases
Sulfatases
Esterases/proteases
Lipases
Phosphatases
-D-Galactosidases
-D- Galactosidases
-D-glucosidases
Indoxyl acetate
Indoxyl phosphate
Indoxyl sulfate
naphtyl acetate/butyrate+FastBlueRR
naphtyl palmitate + ””
naphtyl phosphate + ””
naphtyl -D-galactopyranoside + ””
naphtyl -D- galactopyranoside + ””
naphtyl -D-glucopyranoside + ””
Enzymes and detection systems
The ZAP Express vector allows bouth eukaryoticand prokaryotic expression and accomodates DNA insert from 0 to 12 kb in length.
„Oil“ library = 1,8 x 106 phage particles. Average insert size - 7.5 kbp
Clones in the ZAP Express vector can be screend with either DNAprobes or antibody probes
Phage expression system
Insert cloned into the ZAP Express vector excised out of the phage in the form of the Km-resistant pBK-CMV phagemid vector
Screening of ca. 10000 phage clones yields ca. 20 positives
Excision
Selected clones clustered
500 600 700 800 900 1000 1100
20
40
60
80
100 518.3
569.2598.1
637.2
673.4711.3
775.3
826.0
867.0925.5
994.71035.4
Expression
MALDI-TOF
Purification
Sequencing of selected clones
Product, enzymology
From phage library to enzyme
A short DNA probe anneals to a target DNA of interest. The probe then acts as a primer for a Rolling Circle Amplification reaction
1
The free end of the probe anneals to a small circular DNA template. A DNA polymerase (white oval) is added to extend the primer.
2
The DNA polymerase extends the primer continuously around the circular DNA template generating a long DNA product that consists of many repeated copies of the circle.
34
By the end of the reaction, the polymerase gene-rates many thousands of copies of the circular template, with the chain of copies tethered to the original target DNA. This allows for spatial reso-lution of target and rapid amplification of the signal.
RCA
Polymerase of phage
Perfectly works with small circular templates
Requires improvement of reaction conditions for whole-genomeamplification
Oil enrichment of a selected sample yielded “typical” marine HC-degraders, Marinobacter spp. and pseudomonads
Isolation will be continued with other samples/enrichmentsThose enrichments will be analysed for taxonomy of populations
Conclusions and outlook
The techniques used make possible to detect the enzymatic activities in metagenomes (dozens of clones per single activity screening)
Isolation of a variety of enzymes will be continued
Lack of material. To make a compehensive library, few dozens of micrograms of DNA or some dozens miligrams of the fresh biomass are required. Large-scale samplings of max. 1-2 sites (min 200 L) are needed