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 PlusA-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 PlusO-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 PlusA-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 PlusA-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 PlusA-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 PlusA-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 PlusA-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 PlusB-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 tagSUBSTART = 37 means n-1 truncationSUBSTART = 38 means n-2 truncationSUBSTART >> 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