Strategies and Results of Routine Semi-automated Cloning

Heath Klock - JCSG

Our Vector Strategy

• Small, non-cleavable expression/purification tag directly followed by the gene of interest

• Blunt/Pac strategy directs orientation while eliminating extra codons/residues in the N-terminal tag

• Toxic ccdB protein product is expressed in transformants that contain undigested or religated vector background

Para

6-aa

Pml I

6-hisCAC GTG Cm/ccdB TTA ATT AA

Pac I

Pme I

Nco I

RBSSma I

PT7

Our Oligo Strategy

• 5’ Primer– Begins with the first codon of the reading frame to be

expressed

• 3’ Primer – Contains a stop codon, an insert-specific 6 base

“SYBR” sequence, and a Pac I restriction site

• Strategy allows for the same PCR product to be ligated into various Blunt/Pac-adapted vectors

Gene Product of Interest

Stop

SY

BR

Pac I

Our robotics…

• MWG Biotech - Roboseq 4204 SE– Liquid and plate handling

for PCRs, clean-ups, digests and ligations

• Bio-tek - Synergy HT – Microplate reader for

absorbance and fluorescence, kinetic and endpoint assays

Two more pieces…

• Primus HT – Multi-block thermocyclers– Motorized lids

– PCRs, digests and ligations

• ST Robotics – R16 robot arm – Transfers plates between

main deck, plate reader, thermocyclers and off-deck storage

The robotics have been integrated to create “Tabitha”

R16armRoboSeq

Liquid Handling

Deck

SynergyHT

PlateReader

Primus HT

Thermo cyclers

Off deckPlateStorage

6 ft.

6 ft.

Process and Timeline for 96 Targets• Day 1

– Initial PCR setup– Gel Analysis*– PCR Cleanup – Pac I digest– Ligations– Transformations

• Day 2– Colony Picking

• Day 3– Diagnostic PCR setup*– SYBR assay*– Reracking positives*– Exo/SAP– Sequence

• Later– Analyze sequence*– Final glycerol stocking

Stagger start “Day 1” up to 4 times per week for attempting ~384 targets in a week

not currently automated

Workload requirements per plate of targets

Final glycerol stock archive (Later)

Rerack, Exo/SAP, sequence (Day Three)

Colony picking (Day Two), dPCR, SYBR (Day Three)

Ligation, transformation (Day One)

PCR, cleanup, Pac digest (Day One)

Gel Loading and Documentation by Tabitha and E-gels

• Samples of completed PCRs are loaded onto an E-gel 96 (Invitrogen) along with a DNA ladder

• E-gel 96 Editor (Invitrogen) converts the raw image into a well-labeled, notebook-ready image

• Cells from cultures of isolated colonies are used directly as template for automated dPCR reaction setup. No minipreps!!

• A 5’ vector-specific primer is used with a 3’ primer that is universally specific to insert-containing plasmids only

• PCR works only when an insert is present

Diagnostic PCR

GOI

No GOI

Insert present.

PCR product accumulates!!

No insert.

No PCR product.

SYBR Assays and Amplicon Reracking

• Fluorescence intensity of SYBR Gold is enhanced when bound to double-stranded PCR product vs. single-stranded oligonucleotides

• Wells with accumulated PCR product (vector with insert) fluoresce ~10-fold more intensely than those wells without PCR product (vector without insert)

• An Excel spreadsheet identifies the two most fluorescent SYBR-positive wells for each putative clone and creates rerack files for consolidating amplicons for Exo/SAP reactions and submission to sequencing

• 80-90% correlation to running gels. 10 times faster!!!

Sequence Analysis• Reracked PCR products are

submitted to sequencing. Again, no minipreps!!

• ABI sequence trace files are converted to FastA files by Seqman (DNAStar)

• FastA sequence files are uploaded into CloneCompare (GNF)

• CloneCompare finds the 5’ 6thio/6his tag, BLASTs the adjacent, downstream sequence against a pre-compiled database of clone candidates and returns the clone’s identity as well as n-1 truncation information

GRADE Well Location Accession ID% MATCHLEN QLEN Gene Length QSTART SUBSTART STRANDA-Good 'M4rr1.pBADF_A01_A01_001.ab1' BC019519 98 676 823 1158 140 36 Plus

A-Good 'M4rr1.pBADF_A02_A02_005.ab1' BC019812 98 626 776 1017 140 36 PlusA-Good 'M4rr1.pBADF_A03_A03_017.ab1' BC019856 99 544 687 1359 140 36 Plus

O-Unclassified 'M4rr1.pBADF_A07_A07_049.ab1' BC020180 98 682 846 3201 155 2314 PlusA-Good 'M4rr1.pBADF_A08_A08_053.ab1' BC021159 99 683 833 1119 143 36 Plus

A-Good 'M4rr1.pBADF_A09_A09_065.ab1' BC021466 98 643 792 1092 143 36 PlusA-Good 'M4rr1.pBADF_A10_A10_069.ab1' BC019812 99 636 783 1017 143 36 Plus

A-Good 'M4rr1.pBADF_A11_A11_081.ab1' BC019856 98 628 781 1359 146 36 PlusA-Good 'M4rr1.pBADF_B01_B01_009.ab1' BC020046 98 622 769 846 138 36 Plus

A-Good 'M4rr1.pBADF_B02_B02_013.ab1' BC020074 98 676 832 1347 140 36 PlusO-Unclassified 'M4rr1.pBADF_B07_B07_057.ab1' BC019812 99 610 799 1017 143 406 Plus

A-Good 'M4rr1.pBADF_B08_B08_061.ab1' BC019856 99 593 747 1359 144 36 PlusA-Good 'M4rr1.pBADF_B09_B09_073.ab1' BC019453 99 520 760 555 147 36 Plus

A-Good 'M4rr1.pBADF_B10_B10_077.ab1' BC020028 99 651 867 687 143 36 PlusB-Ins/Del 'M4rr1.pBADF_D08_D08_062.ab1' BC020046 98 707 856 846 140 38 Plus

B-Ins/Del 'M4rr1.pBADF_G01_G01_004.ab1' BC021598 99 599 745 759 139 39 Plus

SUBSTART = 36 means the gene it identifies starts perfectly right after the 6thio/6his tag

SUBSTART = 37 means n-1 truncationSUBSTART = 38 means n-2 truncation

SUBSTART >> 36 means large truncation

6thio/6his Gene of Interest

CloneCompare

Cloning Results to Date

T. maritimaTma Orthologs

(in process)Mouse

(in process)

Targets 1877 1070 400

Amplifers 1800 799 287

%Amplified 96% 75% 72%

Clones 1777 564 256

% of Amplifers Cloned 99% 71% 89%

% of Targets Cloned 95% 53% 64%

Conclusions

• Developed a highly, not fully, automated platform for cloning

• Requires no minipreps and only one DNA gel of 96 samples for every 480 PCR reactions

• The use of SYBR assays and CloneCompare facilitates fast, “hands-free” identification of expression-ready clones

• Large numbers of clones can be generated quickly with success rates similar to manual cloning using only one person’s time

Acknowledgements

• Aprilfawn White– Tabitha specialist

• Tanya Shin– Cloning

• Scott Lesley– Program Head

• Tony Orth– Cloning Strategies

• Keith Ching– Sequence Analysis

• Michael Winniman– MWG robotics

integration

• NIH PSI P50 GM62411