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%!

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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!

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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=;

I! I! I!I! I! I!I! I! I!I! I! I!I! I! I!I! I! I!

J++%!

J++%!

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J++%! J++%! J++%!

J++%!

!"#"$%"& !"#$#!%&''() *!+,!-%+.(/ 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=;

I! I! I!I! I! I!I! I! I!I! I! I!I! I! I!I! I! I!

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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