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Fosmids and functional screening
11-‐29-‐2011
Mari Nyyssonen
DOE Project Meeting, UC Irvine
Sample pooling for fosmid libraries
x 4
Sample pooling for fosmid libraries !"#"$%"& !"#$#!%&''() '"%&()&* !"#$$!%&''() +(," !"#$$!%&''() -"./"$%"&!"#$$!%&''()*#$+,, *#$+,, *#$+,, *#$+,,*#-.,, *#-.,, *#-.,, *#-.,,*$/+,, *$/+,, *$/+,, *$/+,,*$0+,, *$0+,, *$0+,, *$0+,,*#0.,, *#0.,, *#0.,, *#0.,,*$"+,, *$"+,, *$"+,, *$"+,,*$1.,, *$1.,, *$1.,, *$1.,,*"".,, *"".,, *"".,, *"".,,*#2.+, *#2.+, *#2.+, *#2.+,*#3++, *#3++, *#3++, *#3++,*#4.+, *#4.+, *#4.+, *#4.+,*$$++, *$$++, *$$++, *$$++,*$-++, *$-++, *$-++, *$-++,*$3++, *$3++, *$3++, *$3++,*"#.+, *"#.+, *"#.+, *"#.+,*"$.+, *"$.+, *"$.+, *"$.+,*#$.,5 *#$.,5 *#$.,5 *#$.,5*#-+,5 *#-+,5 *#-+,5 *#-+,5*#0+,5 *#0+,5 *#0+,5 *#0+,5*$".,5 *$".,5 *$".,5 *$".,5*$/.,5 *$/.,5 *$/.,5 *$/.,5*$0.,5 *$0.,5 *$0.,5 *$0.,5*$1+,5 *$1+,5 *$1+,5 *$1+,5*""+,5 *""+,5 *""+,5 *""+,5
6! 6! 6! 6!6! 6! 6! 6!6! 6! 6! 6!6! 6! 6! 6!6! 6! 6! 6!6! 6! 6! 6!
788%!
788%!
788%!
788%!
788%!
788%!
788%!
788%!
788%!
788%!
788%!
788%!
788%!
788%!
788%!
788%!
788%!
788%!
788%!
788%! 788%! 788%!
788%! 788%!
788%!
788%!
788%!
788%!
788%!
788%!
788%!
HMW DNA cloning
0.8 % agarose in 0.5 x TBE, 35V, 18 h, 4°C 0.8 % E-‐Gel, 5 min, λ DNA standard
100 ng
75 ng
50 ng
25 ng
10 ng
Dec
_10
Feb_
11
Sep_
11
Dec_10 Feb_11 Sep_11
36 kb
10 kb
48 kb 36 kb
10 kb
48 kb
CopyControl Fosmid Library Production Kit (Epicentre)
Fosmid libraries !"#"$%"& !"#$#!%&''() *!+,!-%+.(/ '"%&()&* !"#$$!%&''() *!+,!-%+.(/ +(," !"#$$!%&''() *!+,!-%+.(/ -"./"$%"&!"#$$!%&''() *!+,!-%+.(/0#$122 0#$122 0#$122 0#$1220#3422 5#### 0#3422 "$6### 0#3422 73## 0#3422 568##0$8122 9"$:55; 0$8122 0$8122 0$81220$<122 0$<122 0$<122 0$<1220#<422 0#<422 0#<422 0#<4220$"122 =56### 0$"122 356### 0$"122 $6>5# 0$"122 :##0$>422 0$>422 0$>422 0$>4220""422 0""422 0""422 0""4220#=412 0#=412 0#=412 0#=4120#5112 ":6### 0#5112 $:6### 0#5112 73## 0#5112 ?@A40#:412 9"">>#; 0#:412 0#:412 35# 0#:4120$$112 0$$112 0$$112 0$$1120$3112 0$3112 0$3112 0$31120$5112 ?@A4 0$5112 3:6### 0$5112 0$51120"#412 0"#412 0"#412 0"#4120"$412 0"$412 0"$412 0"$4120#$42B 0#$42B 0#$42B 0#$42B0#312B ?@A4 0#312B ?@A4 0#312B 3## 0#312B0#<12B 0#<12B 0#<12B 0#<12B0$"42B 0$"42B 0$"42B 0$"42B0$842B 0$842B 0$842B 0$842B0$<42B ?@A4 0$<42B "=6### 0$<42B 0$<42B0$>12B 0$>12B 0$>12B 0$>12B0""12B 0""12B 0""12B 0""12B
C! C! C! C!C! C! C! C!
C! C! C! C!C! C! C! C!
C! C! C! C!C! C! C! C!
D++%!
D++%!
D++%!
D++%!
D++%!
D++%!
D++%!
D++%!
D++%!
D++%!
D++%!
D++%!
D++%!
D++%!
D++%!
D++%!
D++%!
D++%!
D++%!
D++%! D++%! D++%!
D++%! D++%!
D++%!
D++%!
D++%!
D++%!
D++%!
D++%!
D++%!
HMW DNA isolation from June 2011 litter
• 0.4 g of litter/replicate extraction
• Homogenize litter on TissueLyser at 25 Hz for 30 s (liquid N2 and dry ice)
• Extraction buffer (100 m sodium phosphate, Tris-‐HCl, EDTA, 1.5 M NaCl
and 1 % CTAB)
• Enzymatic and detergent lysis with lysozyme at 37°C for 30 min followed
by proteinase K and SDS at 55°C for 45 min
• Hot phenol extraction with PCIAA (25:24:1) at 60°C for 30 min
• O/n precipitation with PEG at RT
• 18 h gel electrophoresis on 0.8 % agarose and 0.5 X TBE at 35 V and 4°C
• Electroelution at 100 V and 4°C for 90 min
HMW DNA from June samples
0.8 % agarose in 0.5 x TBE, 35V, 18 h, 4°C 0.8 % E-‐Gel, 5 min, λ DNA standard
After electroelution
After end-‐repair
36 kb
10 kb
48 kb 36 kb
10 kb
48 kb 100 ng
75 ng
50 ng
25 ng
10 ng
June
_01X
X June
_08X
X June
_14R
X June
_16X
N
100 ng
50 ng
25 ng
10 ng
June
_01X
X June
_08X
X June
_14R
X June
_16X
N
June_01RX June_08XX June14RX June_16XN
Fosmid libraries
!"#"$%"& !"#$#!%&''() *!+,!-%+.(/ 01!+,!234 '"%&()&* !"#$$!%&''() *!+,!-%+.(/ 01!+,!234 +(," !"#$$!%&''() *!+,!-%+.(/ 01!,!2345#$677 5#$677 5#$6775#8977 :#### ;8# 5#8977 "$<### ;;; 5#8977 =;<### >#?"5$=677 @"$;::A @>8B1A 5$=677 @>;B1A 5$=677 @8=B1A5$?677 5$?677 5$?6775#?977 5#?977 5#?9775$"677 >:<### 5$"677 8:<### 5$"677 ;:<###5$C977 5$C977 5$C9775""977 5""977 5""9775#>967 5#>967 5#>9675#:667 ";<### 5#:667 $;<### 5#:667 =#<###5#;967 @""CC#A 5#;967 5#;9675$$667 5$$667 5$$6675$8667 5$8667 5$86675$:667 D4E9 5$:667 8;<### 5$:667 ?#<###5"#967 5"#967 5"#9675"$967 5"$967 5"$9675#$973 5#$973 5#$9735#8673 D4E9 5#8673 D4E9 5#8673 ;:<###5#?673 5#?673 5#?6735$"973 5$"973 5$"9735$=973 5$=973 5$=9735$?973 D4E9 5$?973 "><### ?"? 5$?973 =?<###5$C673 5$C673 @>=B1A 5$C6735""673 5""673 5""673
;F8GH#? ;>CF=;
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!"#"$%"& !"#$#!%&''() *!+,!-%+.(/ 01!+,!234 '"%&()&* !"#$$!%&''() *!+,!-%+.(/ 01!+,!234 +(," !"#$$!%&''() *!+,!-%+.(/ 01!,!2345#$677 5#$677 5#$6775#8977 :#### ;8# 5#8977 "$<### ;;; 5#8977 =;<### >#?"5$=677 @"$;::A @>8B1A 5$=677 @>;B1A 5$=677 @8=B1A5$?677 5$?677 5$?6775#?977 5#?977 5#?9775$"677 >:<### 5$"677 8:<### 5$"677 ;:<###5$C977 5$C977 5$C9775""977 5""977 5""9775#>967 5#>967 5#>9675#:667 ";<### 5#:667 $;<### 5#:667 =#<###5#;967 @""CC#A 5#;967 5#;9675$$667 5$$667 5$$6675$8667 5$8667 5$86675$:667 D4E9 5$:667 8;<### 5$:667 ?#<###5"#967 5"#967 5"#9675"$967 5"$967 5"$9675#$973 5#$973 5#$9735#8673 D4E9 5#8673 D4E9 5#8673 ;:<###5#?673 5#?673 5#?6735$"973 5$"973 5$"9735$=973 5$=973 5$=9735$?973 D4E9 5$?973 "><### ?"? 5$?973 =?<###5$C673 5$C673 @>=B1A 5$C6735""673 5""673 5""673
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Library coverage
!"#
$!"#
%!"#
&!"#
'!"#
(!!"#
)*+,!(--#.(/0# )*+,!'--#.$$0# 1*2,!(--#.('0# 1*2,!'--#.$/0# 1*2,(&-3#.('0# 456,!(--#.$70# 456,!78-#.$!0# 456,!(-3#.(&0#
9:;<=>*=2?+>*<@?#
A:;<=>*=2?+>*<@?#
B:;<=>*=2?+>*<@?#
C:;<=>*=2?+>*<@?#
D+E6=2?+>*<@F?*#
G?+>*<=@F*>*H#
IJK=<=L*M?K*H#
IK=H><@F@?#
156N@#
O@<@F@PK?6>?*#
Q>J*<#R5S?<T=>*H#
U6+K?HH@V*F#
Estimate of leaf litter community composition by BLAST alignment of illumina reads to Silva LSU-rRNA database
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
leaf litter lane 1
(n = 4,175)
leaf litter lane 2
(n = 6,953)
leaf litter lane 3
(n = 66,136)
leaf litter lane 4
(n = 53,364)
leaf litter lane 5
(n = 15,461)
leaf litter lane 6
(n = 20,541)
Cow rumen (n = 7,856)
Perc
enta
ge o
f hits
to S
ilva
LSU
-rR
NA
Plants
Fungi
Other Eukaryotes
Bacteria
Expression host
Gene Protein Function
phoA Alkaline phosphatase Dephosphorylation (non-‐specific)
aphA Acid phosphatase/phosphotransferase Dephosphorylation (non-‐specific)
appA Phosphoanhydride phosphorylase Dephosphorylation
amyA Cytoplasmic alpha-‐amylase 1,4-‐alpha-‐D-‐glucan degradation
malS Periplasmic plasmic alpha-‐amylase 1,4-‐alpha-‐D-‐glucan degradation
malZ Maltodextrin glucosidase 1,4-‐alpha-‐D-‐glucan degradation
yihQ Alpha-‐glucosidase 1,4-‐alpha-‐D-‐glucan degradation
EPI300-‐T1R (F-‐ mcrA Δ(mrr-‐hsdRMS-‐mcrBC) (StrR) ϕ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7679 galU galK λ-‐ rpsL nupG trfA tonA dhfr)
0% 20% 40% 60% 80% 100%
February 2011 (22)
December 2010 (19) α-‐Proteobacteria
β-‐Proteobacteria
γ-‐Proteobacteria
Actinobacteridae
Chloroflexales
Clostridia
Bacteroidetes
Fungi
Viridiplantae
Unclassified
Starch degradation Alkaline phosphatase
Expression host
Functional HTP screening • Chromogenic and fluorescent substrates
• Degradation pathways
Cellulose Cellobiose β-‐D-‐glucoside
Function Enzyme Substrate Substrate concentration
Substrate added
Detection
Assay type Assay duration (d)
Cellulose degradation
Endo-‐cellulase Cellobiohydrolase β-‐glucosidase
AZCL-‐cellulose 4-‐MUB-‐β-‐D-‐cellobioside PNP-‐β-‐D-‐glucoside
0.1 % (w/v) 1 mM 1 mM
In the beginning In the beginning In the beginning
590 nm 365/450 nm 410 nm
96 well plate 5
Hemicellulose degradation
Endo-‐xylanase β-‐xylosidase
AZCL-‐xylan 4-‐MUB-‐β-‐D-‐xyloside
0.1 % (w/v) 1 mM
In the beginning In the beginning
590 nm 365/450 nm
96 well plate
5
Starch degradation
α-‐amylase α-‐glucosidase
Starch Azure 4-‐MUB-‐α-‐D-‐glucoside
0.5 % (w/v) 1 mM
In the beginning In the beginning
600 nm 365/450 nm
96 well plate 5
Chitin degradation
Chitinase N-‐acetyl-‐glucosaminidase
Chitin azure 4-‐MUB-‐β-‐N-‐acetyl-‐β-‐glucosamide
0.5 % (w/v) 1 mM
In the beginning In the beginning
590 nm 365/450 nm
96 well plate 5
Peptide breakdown
Protease Skim milk 2 % In the beginning Visual Bioassay plate 5
PO43-‐ release
from organic sources
Phosphatase BCIP 40 μg/ml In the beginning
Visual Bioassay plate 3
Lignin degradation
Polyphenol oxidase L-‐dihydroxyphenyl alanin/Syringaldazine
5 mM After o/n incubation
Absorbance 96 well plate 5
Plating primary library on selective media
Picking of individual clones on 96 well plates
37°C o/n QFill3, Qpix2
Store parent plates at -‐80°C
SCREENING
Array clones on bioassay plates containing screening substrate
Incubate at 37°C for 2-‐5 days
Visual inspection, manual picking and re-‐screening of positives
…………… ………….. ………….. ………..
Fill plates with growth medium
supplemented with screening substrate
(fluorescent or colorimetric) Pool clones from parent
plates to 96 deep well plates
Incubate at 37°C for 2 to 5 days
Spin down biomass and scan
(Automated) Picking and re-‐screening of positives
QFill3/Manual
Biomek FX
QPix2
LIMS
Screening results
• Plant litter collected on December 2010 from
control plot
• 21,755 clones picked with QPix2
• 12,160 clones (0.4 Gb) screened against 8 substrates with Biomek FX and FXp
Multiplexed detection
√
A B
C D
E F
G H
0
2
4
6
1 2 3 4 5 6 7 8 9 10 11 12
Abs
orba
nce
(10-1
)
A B
C D
E F
G H
0
2
4
6
1 2 3 4 5 6 7 8 9 10 11 12
Fluo
resc
ence
(108 )
α-‐amylase α-‐glucosidase
Function Substrate Positives Hit rate (per clones)
Hit rate (per bp)
Cellulose degradation
AZCL-‐cellulose 4-‐MUB-‐β-‐D-‐cellobioside PNP-‐β-‐D-‐glucoside
-‐ 7 3
-‐ 1,737 4,053
-‐ 57,142 133,333
Hemicellulose degradation
AZCL-‐xylan 4-‐MUB-‐β-‐D-‐xyloside
-‐ 12
-‐ 1,013
-‐ 33,333
Starch degradation
Starch Azure 4-‐MUB-‐α-‐D-‐glucoside
3 9
4,053 1,351
133,333 44,444
Chitin degradation
Chitin azure 4-‐MUB-‐β-‐N-‐acetyl-‐β-‐glucosamide
-‐ 6
-‐ 2,027
-‐ 66,667
Lignin degradation
L-‐dihydroxyphenyl alanine Syringaldazine
-‐ -‐
-‐ -‐
-‐ -‐
Positive clones -‐ screening
Positive clones – end-‐sequencing
• Alpha amylase (Actinobacteria, Dec_01XX)
• Alkaline phosphatase (Bacteroidetes, Dec_08XX)
• Putative glycosidase (Alpha-‐Proteobacteria, Dec_08XX)
• Xylosidase/arabinosidase/GH 43 (Bacteroidetes, June_01XN)
Full length cDNA libraries -‐ cDNA capture -‐
• Eukaryotic mRNA • 3’ poly-‐A tail • 5’ cap
Full length cDNA libraries -‐ cloning and expression -‐
Bacterial expression Yeast expression
?
Full length cDNA libraries -‐ Trichoderma reesei RUT-‐30 -‐
• Grow on CMC media to
activate cellulose
degradation
• Isolate total RNA
• Capture full length mRNA
• Clone to Gateway
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