Clone the Unclonable – Vectors and Cells to Capture and Express Problematic DNAs Ron Godiska, Ph.D. May, 2016 Agenda Improve Cloning and Expression of Problematic DNA • What makes DNA difficult to clone or express? • Circular vectors for difficult DNAs • Linear vectors for “impossible” DNAs • Enzyme-free cloning and compatible protein expression vectors • E. coli strains for toxic recombinant proteins or unstable DNA What is Unclonable DNA? Characteristics of Difficult DNA • Toxic coding sequences • Promoters • A-T Rich DNA • Large fragments (>10 kb) • Trace amounts Which types of difficult DNA have you worked with in the lab? Common Vector Traits Cause Cloning Problems Vector driven transcription into the insert – Deleterious expression – Destabilize 2o structure Insert driven transcription out into the vector – False negatives (blue or lethal) – Transcriptional interference pUC19 – Replication interference High copy number – Increased effect of the above issues – Active origin – Many copies/cell Supercoiling – Deletion of secondary structures CloneSmart® Technology “Silencing” DNA Inserts to Improve Cloning • No Transcriptional Interference – Stably maintain “unclonable” DNA • No blue/white screening – No false positives/negatives • Minimal vector size • Variable copy number • Variety of drug selection markers • Very low empty-vector background U.S. Patent 6,709,861 pSMART Vectors Clone Toxic Inserts and Promoters Test DNA Fragments: • RNase coding sequence (350 bp; no promoter) • Phage Lambda PR Promoter (400 bp) pSMART Vectors cDNA Clones are More Stable Deletion Rates: <1% in pSMART HC Kan vs. 32% in pUC % Deleted Clones Insert Size (kb) • Teleost fish cDNA library • Clones were grown in liquid medium, diluted, and regrown • Each final culture was diluted, plated, and grown up into individual colonies • Plasmid DNA was isolated from colonies for each clone and analyzed - Oleksiak M.F., Crawford D.L., 2001. (unpublished) pSMART Vectors Clone A/T-rich Fragments pSMART Vectors Clone A/T-rich Fragments Cloning Target: 2-4 kb Lactobacillus helveticus (65% AT) fragments pSMART LC Kan Vector pUC19 Vector Undigested DNA from transformants pSMART Vectors Clone Large Fragments Cloning target: 8-14 kb Shigella genomic DNA (50% GC) pSMART LC Kan M 1 2 3 4 5 6 7 8 9 M Undigested DNA from transformants pSMART BAC Vectors Clone Large, Difficult DNA Fragments ▪ Transcription-free ▪ Inducible copy number • Single copy • 20-50 copies per cell (oriV) ▪ No blue/white screening pSMART BAC Vectors Clone Large, Difficult DNA Fragments Cloning: Tetrahymena (75% AT) 10-20 kb genomic DNA inserts pSMART BAC Blue/white BAC vector Undigested DNA from transformants BigEasy® Linear Cloning System for Bacterial Cloning Based on pJAZZ® Linear Vectors BigEasy® Linear Cloning System (pJAZZ® Vector) Unmatched Cloning of Difficult Inserts • Unique linear cloning plasmid (from phage N15) • No supercoiling • No circular intermediates • No vector-driven transcription / translation • Low copy number; Inducible to mid copy number • Minimal size bias (up to ~40 kb) • Chloramphenicol or Kanamycin resistance Unmatched cloning capabilities BigEasy® Linear Cloning System Replication of the Linear pJAZZ Vector Ravin et al., NAR v.31 (2003) BigEasy® Linear Cloning System Utilize Standard Cloning Procedures • Standard ligation methods • Standard electroporation • Standard alkaline lysis DNA preps BigEasy® Linear Cloning System Large AT-rich Fragments pJAZZ 10-20 kb inserts of V L.helv. are stable in pJAZZ (NotI digest) pUC19 2-4 kb inserts of L.helv. are unstable in pUC19 (Uncut plasmid DNA) BigEasy® Linear Cloning System AT-rich Fragments Up to 40 kb Tetrahymena (70% AT) Nematode (62% AT) “TIGR had tried several times, but the largest size we could obtain was 4-6 kb.” - Dr. Robert Coyne (J. Craig Venter Institute) BigEasy® Linear Cloning System Extremely AT-rich Fragments Plasmodium berghi genomic DNA (70-90% AT) Plasmodium Genetic Modification Project (PlasmoGem) ~104 genomic clones 4-20 kb inserts (average = 8.7 kb) “These data were provided by the PlasmoGEM group at the Wellcome Trust Sanger Institute and can be obtained from http://plasmogem.sanger.ac.uk/.” Pfander et al., (2011). Nature Methods. 8(12): 1078–1082. BigEasy® Linear Cloning System 100% GC, Fragile X Repeats - (GCC)300 pJAZZ arms pUC19 (GCC)300 ΔGCC BigEasy® Linear Cloning System Unstable, Repetitive DNA Inserts • Highly repetitive mollusk cDNA in pJAZZ OC vector (0.7 - 2 kb inserts. NotI digest) BigEasy® Linear Cloning System Unstable, Repetitive DNA Repeat Sequences White – ATTCT Orange – ATTTTCT Blue – ATATTCT Green – ATTCTTCT Aqua – ATTCC Black – Misc. Human SCA10 genomic repeats cloned into the pJAZZ vector. • Clone “A”: 830 copies (4.4 kb) • Clone “B”: 788 copies (4 kb) • Each box represents one repeat unit McFarland et al., (2015). PLoS One. 10(8):e0135906. pJAZZ® Rham Expression Vector Bacterial Expression of Large Inserts/Operons • Rhamnose promoter for inducible expression • Expression of isoprene from an 8-gene bacterial operon was 10X higher in pJAZZ vs. 2-3 co-transformed circular plasmids PRhm RBS pJAZZ Rhm is available by custom quote. Contact us at [email protected] SUMMARY - BigEasy® Linear Cloning System Extremely stable cloning of Difficult DNA: • AT-rich and GC-rich DNAs (96% AT → 100% GC) • Highly repetitive sequences • Large fragments (up to 40 kb) • Multiple genes • Large inverted or tandem repeats SUMMARY: Bacterial Cloning Vectors Systems for Any Insert Size Options available: • CloneSmart® kits for <15 kb • BigEasy® v2.0 kits for exceptionally difficult targets • CopyRight® kits for Fosmid or BAC cloning Convenient, fast, easy, and effective • Pre-cut, dephosphorylated – ready to clone • <1% Background • Minimize cloning problems Expresso® Vectors for Rapid, Enzyme-Free Cloning Which cloning methods are you currently using? Expresso® Cloning Rapid, Directional, Enzyme-free Cloning • Fast and simple o No PCR purification o No vector preparation o No enzyme incubation step • Seamless and directional • No cloning artifacts or scars • Efficient (>90%) • Multiple tags and promoters available Expresso® Circular Vectors for Protein Expression Rapid, Directional, Enzyme-free Cloning • CloneSmart terminators – prevent transcription into/out of insert • N-terminal or C-terminal 6xHis tags for protein purification • T7 or Rhamnose promoters • Small size (2.2kb) for easier downstream manipulation Expresso® Circular Vectors for Protein Expression Tight Control of Protein Expression with pRham pRham Expresso Vectors Tightly Controlled Expression • Active in K12 or B strains (DH10B, BL21) • Very low background • SUMO or Biotin tags available • Tunable in each cell + Rhamnose M () Expresso® Solubility and Expression System One PCR Product Cloned into 7 Different Vectors • One PCR product (ORF) cloned instantly into 7 different vectors • 7 different solubility fusion tags; one in each vector • Rapidly screen for fusion tags that produce soluble protein Expresso® Solubility and Expression System Solubility Screening of GH1 Protein Fusions Solubility Tags T = Total lysate S = Soluble fraction I = Insoluble fraction • Results: SlyD, Tsf, SUMO, Bla, and MBP demonstrated enhanced solubility (Lane S, ), with minor insoluble band ( ). Vectors for Mammalian Cell Expression Expresso® CMV Cloning and Expression System Mammalian Protein Expression pME-HA Vector: • Circular vector • Minimal size • CMV promoter • CloneSmart terminators • Expresso cloning ➢Fast, easy cloning ➢Expression in mammalian or bacterial cells Expresso® CMV Cloning and Expression System Mammalian or Bacterial Expression from ONE Clone GFP GFP RFIP YFP bGal CHO-K1 COS-7 E. coli Pictures taken 24 hours post-transfection (20x). pJAZZ® Mammalian Expression Vector High Stability Cloning for Mammalian Expression pJAZZ® Mammalian Expression Vector Expression of Large Tandem Repeats pJAZZmamm Subunit I Subunit II Subunit III Subunit IV Right Arm Left Arm • IP3R tetramer gene is 4 x 10 kb, ~1000 Tetramer single ORF. kDa Trimer • Monomers, dimers, and trimers 460 Dimer were expressed from separate kDa constructs. • Tetramer protein (~1000 kDa) is among the largest proteins ever expressed 250 Monomer kDa pJAZZ Mamm is available by custom quote. Alzayady, Godiska, Yule, et al., Contact us at [email protected] J Biol Chem, 2013, 288:29772. pJAZZ® Min Vector Smaller derivative of pJAZZ –OC Expression of Four Genes RFP GFP SEAP β-gal pJAZZ Min is available by custom quote. Contact us at [email protected] Vector Summary Effective, Convenient, Easy, and Quick • pSMART® Circular Vectors: Transcription-free vectors for stable, unbiased cloning of most inserts. • pJAZZ® Linear Vectors: Most powerful cloning vectors ever. • Expresso® Expression Vectors: Most convenient cloning and expression system, with benefits of pSMART vectors. • pJAZZ® Expression Vectors: Bacterial or mammalian expression of extremely difficult targets. Available by custom quote only. • Interactive Selection Guide: Cloning Kits and Vectors http://www.lucigen.com/selection_guide.php?cat=1 E. coli Strains for Difficult DNA Which strains have you used for cloning difficult DNA? OverExpress® C41(DE3) & C43(DE3) Bacterial Cells Expression of Toxic & Membrane Proteins • Mutant strains of BL21 (DE3) for toxic or membrane proteins • Reduced expression of proteins from T7 promoter • Much more robust than pLys strains • Strains may differ in tolerance for various proteins • Available in chemically competent and electrocompetent formats OverExpress® C41(DE3) & C43(DE3) Bacterial