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High Throughput Cloning and Expression of NESG Targets

Jan 2006

Dongyan Wang

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Cloning and Expression Procedures (1)

*Design and order primers

*PCR& Gel extraction

Restriction Digestions

Digestion cleanup

Drying and filtration

Restriction Digestion

*Ligation

Transformation into XL-Gold cells

*Colony screeningPCR & gel

@Miniprep(Archive DNA and GS)

*Miniprep culture

* : Data entry into Excel spreadsheet@ : Enter target set into Platemaster : Steps involving Robot

Fragments cloned into vectors

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Transformation into Magik cells

Take gel pictures (or scan and upload) and score.

*Run samples on SDS-PAGE gels, stain and de-stain.

IPTG induction

Harvest, sonication, and sample preparation

MJ9 culture and dilution

@Expression LB culture(Archive GS)

Upload to Spine

Competition analysis and decision making

*Data entry @Verify Archive

*Transfer to fermentation

Cloning and Expression Procedures (2)

Fragments cloned into vectors

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Work Flow of a Single Process

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>ER84 atgtcccgagtctgccaagttactggcaagcgtccggtgaccggtaacaaccgttcccacgcactgaacgcgactaaacgccgtttcctgccgaacctgc actctcaccgtttctgggttgagagcgagaagcgttttgtcaccctgcgc gtatctgctaaaggtatgcgtgtaatcgataaaaaaggcatcgatacagt

tctggctgaactgcgtgcccgtggcgaaaagtactaa

Primers for Target sets-2 weeks

Target sequence in FASTA format:

Copy & Paste

Primer seq. output

SR430,pET 21-23C,F,NdeI,66,34,172,,ATCGATCGCATATGATGAGCCGCTATGCAAAATG

SR430,pET 21-23C,R,XhoI,66,36,172,,TGACTCTCGAGTATAATACTCTTCCATTTGTTTCCC

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A1 SR430,pET 21-23C,F,NdeI,66,34,172,,ATCGATCGCATATGATGAGCCGCTATGCAAAATG

Well Target Plasmid Dir Sequence Size(bp)Ext RE Int RE

A1 SR430 pET 21-23C F ATCGATCGCATATGATGAGCCGCTATGCAAAATG 172 NdeI none

B1 SR482 pET 21-23C F ATCGATCGCATATGTTGAACATTGAAAGGCTCACTAC 352 NdeI HindIII

Primer data copied from Primer pri’mer is pasted into Excel worksheet:

Primer data parsed into different fields

Well TargetSize (bp)

MW (KD) Ext RE Site

Internal RE Sites Vector PCR Lig Dig

A1 SR430 172 7.1 NdeI/XhoI none 21-23C BamHI

B1 SR482 352 13.7 NdeI/XhoI HindIII 21-23C BamHI

Target data entered into Set Summary Worksheet

Same data used to order primer synthesis in 96-well format from Operon

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1 set = 96 targets

PCR and purification of target fragments (1)Day 1-2

96 pairs of gene-specific primers

And

Genomic DNA template

96 PCR reactionsPCR products separated

On 2% agarose gel

Well TargetSize (bp)

MW (KD) Ext RE Site

Internal RE Sites Vector PCR Lig Dig

A1 SR430 172 7.1 NdeI/XhoI none 21-23C yes BamHI

B1 SR482 352 13.7 NdeI/XhoI HindIII 21-23C no BamHI

Results entered into Excel worksheet

Gel pic storage

Comment field:reason/size

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PCR and purification of target fragments (2)

Day 1-2

Bands of right sizes are manually cut and put into a

96-well block. Gel slices melted at 55 C.Automated gel extraction using Qiagen robot.

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Problems that may arise

Some organism’s genome is GC rich. Requires GC-

rich PCR kit and longer elongation times.No PCR products or wrong size.Robot malfunctions.

Target sequence GC%

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Day 2-3

5’ and 3’ restriction endonuclease digestions for

directional cloning.2 overnight 37 C reactions.

Restriction Digestions

Well TargetSize (bp)

MW (KD) Ext RE Site

Internal RE Sites Vector PCR Lig Dig

A1 SR430 172 7.1 NdeI/XhoI none 21-23C yes BamHI

B1 SR482 352 13.7 BamHI/XhoI HindIII 21-23C no EcoRII

Graphic tools for helping locating different RE/Lig wells

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Cleaning and filtrationDay 4

Automated digestion cleaning up using Qiagen

robot.Lyophilize the plate in speed vacuum till dry.Resuspend in dH2O.Purify DNA using 96-well Centri-Sep plate.

Explore non Centri-Sep methods

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LigationDay 4-5

Ligation reaction at 16 C overnight with: Cut and purified target DNA PCR product. Appropriate precut pET vectors. T4 DNA ligase.

65 C 10 min to inactivate the ligase.Ligation digestion at 37 C for 1 hr.

Well TargetSize (bp)

MW (KD) Ext RE Site

Internal RE Sites Vector PCR Lig Dig

A1 SR430 172 7.1 NdeI/XhoI none 21-23C yes BamHI

B1 SR482 352 13.7 BamHI/XhoI HindIII 21-23C no EcoRII

Graphic tools for helping locating different RE/Lig wells

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Transformation into XL-gold

Day 5-6

Mix XL-gold competent cells with digested ligated

DNA on ice. Heat shock at 42 C 1 min.Incubate with SOC 37 C 1 hr.Plate on LB/Amp plates.Incubate overnight 37 C.

96 LB/Amp plates

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Result of transformation into XL-gold

Day 6

Primer Well # of colonies

A1 5

B1 0

C1 20

D1 12

E1 20

F1 20

G1 20

H1 20

Enter the number of colonies of each target into worksheet

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Colony screening (1)Day 6-7

Pick 2 colonies per target, resuspend in water. PCR reactions with T7 primers, which anneals to

vector sequences flanking the inserts.Run PCR products on 2% agarose gel.Select clones with right size.Enter result into Excel worksheets.

WellPrimerWell Target

Clone # Plasmid Full Name

Expected Size (bp)

Right Size?

Picked?

A1 A1 SR430 1 21-23C SR430-21.1 372 no no

B1 A1 SR430 2 21-23C SR430-21.2 372 yes yes

C1 B1 SR482 1 21-23C SR482-21.1 552 yes yes

D1 B1 SR482 2 21-23C SR482-21.2 552 yes yes

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Colony screening (2)

For the targets with only one or no positive clones,

pick more clones from the XL-gold transformation

plate. Repeat colony PCR.Enter result into Excel worksheets.Repeat these steps if needed.

Extra1-2 days

WellPrimerWell

Clone # Plasmid Full Name

Expected Size (bp)

Right Size? Picked?

A1 A1 3 21-23C SR430-21.3 372 no no

B1 A1 4 21-23C SR430-21.4 372 yes yes

C1 E8 3 21-23C SR465-21.3 555 yes yes

D1 E8 4 21-23C SR465-21.4 555 yes yes

192 clones

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Miniprep cultureDay 7-8

Fill out the Miniprep Setup Excel worksheet. Inoculate 10 ul of the right construct into LB/Amp.Shake 37 C overnight.

WellCol

PCR#Col PCR

Well TargetSize (bp)

Size (KD)

Ext RE Sites

Clone # Plasmid Full Name

A7 1 A7 SR478 238 9.53 NdeI/XhoI 1 21-23C SR478-21.1

B7 1 B7 SR478 238 9.53 NdeI/XhoI 2 21-23C SR478-21.2

C7 3 A2 SR423 379 14.7 NdeI/XhoI 3 21-23C SR423-21.3

4 X

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MiniprepDay 8

Obtain archiving labels for the DNA and glycerol

stock of the target set. Make duplicate glycerol stock plates of the miniprep

culture according to the archive SOP.Spin to collect cell pellets.Miniprep using Qiagen robot.Archive DNA plates according to the archive SOP.

Barcode?

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Transformation into expression cells

Day 8-9

Mix 1 ul of miniprep DNA with Magik competent

cells on ice.Heat shock 42 C 1 min.Plate on 24-well LB/Amp/Kan/Glucose plates.Incubate overnight at 37 C.

8 X 24-well platesMiniprep DNA

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Small scale expressionand inductions (1)

Day 9-11

Inoculate 1 colony from the Magik transformation

plate into 0.5 ml LB/Amp/Kan/Glucose.Shake 6 hr 37 C.Make glycerol stock plate of the culture according

to the archive SOP.Inoculate 0.5 ml MJ9 mediaShake overnight at 37 C.

2 Xcolonies

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Small scale expressionand inductions (2)

Day 9-11

Read OD600 of the overnight MJ9 culture. Select a

few wells and dilute 1:10. Inoculate 2 ml MJ9 with the overnight culture so

that the staring OD600 is 0.1 to 0.2..Grow at 37 C until OD600 reaches 0.5 to 0.7.Induce with IPTGShake overnight at 17 C.

2 X 8 X

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Expression proteinsample preparations

Day 11

Spin plates to collect cell pellets.Add Lysis buffer to resuspend cells and keep on ice.Sonicate for 40 min.Save sonicated TOTAL lysate sample (T).Spin to collect SUPERNATANT sample (S).Add protein gel loading buffer to the samples.

2 X 2 X(T) s + (S) s

= 384 samples

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SDS-PAGE gelsDay 12-13

Gel #261 Gel #262

Lane Well Target Name MW Expression SolubilityCompetition

analysis

1 --- MW Ladder --- --- --- ---

2 A7 SR478-21.1 9.5

3 B7 SR478-21.2 9.5

4 C7 SR423-21.3 14.7

5 D7 SR423-21.2 14.7

6 E7 Size? Size?

7 F7 SR501-21.1 15.0

8 G7 SR417-21.1 14.7

9 H7 SR417-21.2 14.7

10 A8 SR461-21.1 9.9

11 B8 SR461-21.2 9.9

12 C8 SR452-21.1 14.8

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SDS-PAGE gelsDay 12-13

Heat samples at 72 C for 10 min.Run samples on SDS-PAGE gels.Wash and stain gels.De-stain gels.Take gel pictures/scan.

384 samples 36 SDS-PAGE gels

96-well Ni-NTA plate?

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Data Management Overview

Analyze SDS-PAGE gels, score expression and

solubility.Enter data into Excel worksheet.Verify that all the wet reagents are archived.Upload cloned constructs and small scale

expression data into SPiNE. Perform competition analysis.Transfer to fermentation.

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Gel #261 Gel #262

Lane Well Target Name MW Expression SolubilityCompetition

analysis

1 --- MW Ladder --- --- --- ---

2 A7 SR478-21.1 9.5 5 5

3 B7 SR478-21.2 9.5 5 5

4 C7 SR423-21.3 14.7 4 3

5 D7 SR423-21.2 14.7 4 3

6 E7 Size? Size?    

7 F7 SR501-21.1 15.0 3.5  0

8 G7 SR417-21.1 14.7 2 2

9 H7 SR417-21.2 14.7 2 2

Expression and Solubility Scores

Gel picture storage

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Spine construct upload

TARGET

NEW ENTR

Y ResearcherINSERT

TYPE VECTOR TAGN-Tag

SequenceC-Tag

Sequence CLONED ON

ER229 -15.1 Dongyan Wang Full Protein pET15 N-HexHis mghhhhhhsh   8/30/2005

ER229 -15.2 Dongyan Wang Full Protein pET15 N-HexHis mghhhhhhsh   8/30/2005

ER228A -15.1 Dongyan Wang Subsequence pET15 N-HexHis mghhhhhhsh   8/30/2005

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Spine small scale expression upload

TARGETNEW

ENTRY Researcher Exp.onHOST

STRAINGrowth. TEMP

Induct.

Temp MEDIAExpr. Scale EXPR PHASE Solubility

SR482-21.1 -ss

Dongyan Wang 7/25/2005

BL21(DE3)+Magic 37 17 MJ9 Analytical 5

soluble+inclusion 5

SR482-21.2 -ss

Dongyan Wang 7/25/2005

BL21(DE3)+Magic 37 17 MJ9 Analytical 5

soluble+inclusion 5

SR490-21.1 -ss

Dongyan Wang 7/25/2005

BL21(DE3)+Magic 37 17 MJ9 Analytical 3

inclusion body 0

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Gel #261 Gel #262

Lane Well Target Name MW Expression SolubilityCompetition

analysis

1 --- MW Ladder --- --- --- ---

2 A7 SR478-21.1 9.5 5 5 selected

3 B7 SR478-21.2 9.5 5 5   selected

4 C7 SR423-21.3 14.7 4 3 purified

5 D7 SR423-21.2 14.7 4 3   purified

6 E7 Size? Size?      

7 F7 SR501-21.1 15.0 3.5  0

8 G7 SR417-21.1 14.7 2 2 cloned

9 H7 SR417-21.2 14.7 2 2  cloned

10 A8 SR461-21.1 9.9 3 3 Crystallized

11 B8 SR461-21.2 9.9 0 0

12 C8 SR452-21.1 14.8 4 4 PDB e-09 

Competition analysis

Link/Buttons

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Transfer to FermentationEnter into Excel worksheet the targets that satisfy

the following criteria:EXS >=8No PDB/PDBH hits or E value >= 0.01.Other criteria?

Transfer to fermentation

WellPrimer

Well TargetOriginal Size

(bp)

Original Size (KD)

Ext RE Sites

Clone # Plasmid Full Name

A7 A4 SR478 238 9.526667 NdeI/XhoI 1 21-23C SR478-21.1

C7 B4 SR423 379 14.69667 NdeI/XhoI 3 21-23C SR423-21.3

G7 D4 SR417 379 14.69667 NdeI/XhoI 1 21-23C SR417-21.1

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Target set statistics

Example Set Result:

Working Step # Failed #Success

Selected 0 96

PCR 3 93

Cloning  2 91

Expression  11 80

Solubility  15 65

Low Solubility  15 50

PDB Hit  5 45

Fermentation    45

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Summary of a target’s life in the NESG cloning pipeline

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Target Selected

PCR success

Cloned Not cloned

Clone #1 Clone #2

Primers designed

PCR failure

Target sequence

Primer data

Ligation well Ligation dateTransformation colony #

•Well locations & Clone #•Col PCR gel pics•Miniprep well locations•Miniprep culture•Archive locations

PCR result, gel picture

Data Recorded at Each Step

Die Step

Success

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Clone of target

Expression failure Expressed

Insoluble Soluble

Low solubility High solubility

E value <=0.01 E value >0.01

Fermentation

•Expression transformation results•Expression date•Archive locations•SDS-PAGE gel setup•SDS-PAGE gel pictures•Gel scores•Competition analysis results•Fermentation list

Data Recorded

Die Step

Success

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It would be nice to have in PLIM

Target data recorded at each working step.Search by target name.Statistics of target results.Graphic tool to help working with 96-well plates. Notes of problems during the processesLink to archiving.Buttons for PDB competition analysis (current e-

mail search).