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 P ara 6-aa Pml I 6-his CAC GTG Cm/ccdB TTA ATT AA Pac I Pme I Nco I RBS Sma I P T7

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 StopSYBR 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” R16 arm RoboSeq Liquid Handling Deck Synergy HT Plate Reader Primus HT Thermo cyclers Off deck Plate Storage 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 6thio/6his Gene of Interest CloneCompare

Cloning Results to Date T. maritima Tma Orthologs (in process) Mouse (in process) Targets Amplifers %Amplified 96%75%72% Clones % 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