Molecular Biology Techniques Supporting IBBE Jared Cartwright Protein Production Lab Head Contact Details: email jared.cartwright@york.ac.uk Phone 01904 328797
Presentation Aims Gene synthesis Cloning PCR based RE cloning In vitro Recombineering LIC / Gateway / Cre-LoxP SLIC Gibson assembly Golden Gate cloning for repetitive DNA sequence Site-directed mutagenesis Gene knockouts E. coli Lambda RED recombinase
DNA Where does it come from? Genomic PCR cdna Gene synthesis completely synthetic double-stranded DNA molecules codon optimised Limitations: secondary structures caused by inverted repeats, extraordinary high or low GC-content, or repetitive structures
Creation of Recombinant DNA Molecules in vitro GAATTC CTTAAG OH DNA I G CTTAA EcoRI and separation of restriction fragments P Ligation OHP Annealing G AATTC CTTAA G P OH GAATTC CTTAAG P DNA II AATTC G GAATTC CTTAAG OH DNA from any source can be joined to give recombinant DNA Cohesive ends joined covalently by DNA Ligase Transformation of recombinant plasmids clones the DNA
DNA Ligase P P P P P P OH B B B B B B B B B B B B B B P P P P P P A P P P P P P P OH B B B B B B B B B B B B B B P P P P P P P P P P P P B B B B B B B B B B B B B B P P P P P P Enzyme-AMP Enzyme AMP Two types of DNA ligase E. coli (NAD + ) T4 Phage (ATP) NAD + and ATP are donors of the AMP
Example: Protein Expression Cloning ATG X Promoter RBS Open Reading Frame Term 5 3 Vector MCS Vector N or C-terminal tag Bacterial Shine-Dalgarno Transcription term Eukaryotic Kozak Poly adenylation
PCR Cloning Use PCR to give specific amplification of gene PCR + Vector Expression construct Design strategy involves: Analysis of target vector for suitable RE sites Analysis of target gene for potentially interfering RE sites Primer design
Target Sequence 5 CACGACCGCTCAGCCAACATGTTTAAGGTAATCACTGTGTGCTTATG <M F K V I T V C L W <--- signal peptide GCTATTCGCCTTCAACAGTCTTAATGTGTCGTCCGAGTATGTTTATGAGGG L F A F N S L N V S S E Y V Y E G --><--mature protein TTTGAAG-------------------------------------------- L K -------------------------------------------- --TACTTACAACCTCAAGAATGAACGAAGATGCATAATCTAATTTATA 3 --V L T T S R M N E D A * -- mature protein-->
PCR Cloning BamHI 5 CTCAGGATCCATGTTTAAGGTAATCACTGTGTG 3 5 CACGACCGCTCAGCCAACATGTTTAAGGTAATCACTGTGTGCTTATG <M F K V I T V C L W GCTATTCGCCTTCAACAGTCTTAATGTGTCGTCCGAGTATGTTTATGAGGG L F A F N S L N V S S E Y V Y E G TTTGAAG-------------------------------------------- L K -------------------------------------------- --TACTTACAACCTCAAGAATGAACGAAGATGCATAATCTAATTTATA 3 --V L T T S R M N E D A * HindIII 3 GTTCTTACTTGCTTCTACGTATTTTCGAACGCG 5
PCR Polymerase Chain Reaction (PCR) (Saiki et al. 1988) ds DNA containing target sequence Add primers and Taq Polymerase Multiple copies
Purified PCR Product 5 CTCAGGATTCATGTTTAAGGTAATCACTGTGTGCTTATG 3 GAGTCCTAGGTACAAATTCCATTAGTGACACACGAATAC GCTATTCGCCTTCAACAGTCTTAATGTGTCGTCCGAGTATGTTTATGAGGG CGATAAGCGGAAGTTGTCAGAATTACACAGCAGGCTCATACAAATACTCCC TTTGAAG-------------------------------------------- AAACTTC-------------------------------------------- --TACTTACAACCTCAAGAATGAACGAAGATGCATAAAAGCTTGCGC 3 --ATGAATGTTGGAGTTCTTACTTGCTTCTACGTATTTTCGAACGCG 5
Digested PCR Product BamHI GATTCATGTTTAAGGTAATCACTGTGTGCTTATG GTACAAATTCCATTAGTGACACACGAATAC GCTATTCGCCTTCAACAGTCTTAATGTGTCGTCCGAGTATGTTTATGAGGG CGATAAGCGGAAGTTGTCAGAATTACACAGCAGGCTCATACAAATACTCCC TTTGAAG-------------------------------------------- AAACTTC-------------------------------------------- --TACTTACAACCTCAAGAATGAACGAAGATGCATAAA --ATGAATGTTGGAGTTCTTACTTGCTTCTACGTATTTTCGA HindIII
Recombinant Vector Product Vector DNA Ligase -GATTCATGTTTAAGGTAATCACTGTGTGCTTATG -GTACAAATTCCATTAGTGACACACGAATAC GCTATTCGCCTTCAACAGTCTTAATGTGTCGTCCGAGTATGTTTATGAGGG CGATAAGCGGAAGTTGTCAGAATTACACAGCAGGCTCATACAAATACTCCC TTTGAAG-------------------------------------------- AAACTTC-------------------------------------------- --TACTTACAACCTCAAGAATGAACGAAGATGCATAAA- --ATGAATGTTGGAGTTCTTACTTGCTTCTACGTATTTTCGA- Ligase Vector DNA
Disadvantages of Restriction Enzyme Cloning Useful restriction enzyme sites can be present in the gene of interest Generally requires agarose-gel purification of digested insert and vector Ligation is an inefficient process
Advanced cloning procedures Ligation Independent Cloning (LIC) Uses T4 DNA polymerase and sequence specific digestion to create long (~10-15 nucleotide) cohesive ends Lambda Phage Site Specific Recombination (Gateway ) Efficiently transfer DNA-fragments between plasmids using a proprietary set of recombination sequences Cre-LoxP Uses Cre recombinase from the bacteriophage P1
SLIC (In-Fusion ) Cloning (Sequence and Ligation Independent Cloning) In-Fusion is a cloning product marketed by Clontech Flexible, high efficiency cloning In vitro cloning reaction Technology uses gene-specific primers with extensions homologous to vector ends In-Fusion enzyme creates single stranded regions of homology and then fuses the PCR product to the vector. Simply transform into E. coli and screen for recombinants
In-Fusion cloning Advantages High efficiency cloning reaction Full flexibility clone into any vector Short primer sequences required Minimal size bias Direct expression cloning Disadvantages Reagent cost
In-Fusion cloning
In-Fusion cloning Other uses Multiple fragment cloning
In-Fusion cloning Other uses Site-directed mutagenesis
In-Fusion cloning SDM cont d
InFusion Deletion Mutagenesis Target gene 5 -GAGGATGATGAGGATGAAGAAGAGATCGAGGTTGAGGAGGAACTCTGCAAGCAGGTGAGGTCCAGAGATATATCCAGAGAGGAG-3 GluAspAspGluAspGluGluGluIleGluValGluGluGluLeuCysLysGlnValArgSerArgAspIleSerArgGluGlu E D D E D E E E I E V E E E L C K Q V R S R D I S R E E DELETION TARGET 5 -GAGGATGATGAGGATGAAGAAGAGATCGAG------------------------GTGAGGTCCAGAGATATATCCAGAGAGGAG-3 GluAspAspGluAspGluGluGluIleGlu------------------------ValArgSerArgAspIleSerArgGluGlu E D D E D E E E I E - - - - - - - - V R S R D I S R E E PRIMER DESIGN ptrc_ciz1_mutf 5 -AGATCGAGGTGAGGTCCAGAGATATATCCAGAGAG-3 5 -GAGGATGATGAGGATGAAGAAGAGATCGAGGTTGAGGAGGAACTCTGCAAGCAGGTGAGGTCCAGAGATATATCCAGAGAGGAG-3 3 -CTCCTACTACTCCTACTTCTTCTCTAGCTCCAACTCCTCCTTGAGACGTTCGTCCACTCCAGGTCTCTATATAGGTCTCTCCTC-5 3 -CCTACTACTCCTACTTCTTCTCTAGCTCCACTCCA ptrc_ciz1_mutr
Target plasmid amplification and InFusion ptrc_ciz1_mutf 5 -AGATCGAGGTGAGGTCCAGAGATATATCCAGAGAG-3 5 -GAGGATGATGAGGATGAAGAAGAGATCGAGGTTGAGGAGGAACTCTGCAAGCAGGTGAGGTCCAGAGATATATCCAGAGAGGAG-3 3 -CTCCTACTACTCCTACTTCTTCTCTAGCTCCAACTCCTCCTTGAGACGTTCGTCCACTCCAGGTCTCTATATAGGTCTCTCCTC-5 3 -CCTACTACTCCTACTTCTTCTCTAGCTCCACTCCA PCR ptrc_ciz1_mutr 5 -GAGGATGATGAGGATGAAGAAGAGATCGAGGTGAGGT-3 3 -CTCCTACTACTCCTACTTCTTCTCTAGCTCCACTCCA-5 5 -GAGGATGATGAGGATGAAGAAG -3 3 -CTCCTACTACTCCTACTTCTTCTCTAGCTCCACTCCA-5 5 -AGATCGAGGTGAGGTCCAGAGATATATCCAGAGAGGAG-3 3 -TCTAGCTCCACTCCAGGTCTCTATATAGGTCTCTCCTC-5 InFusion 5 -AGATCGAGGTGAGGTCCAGAGATATATCCAGAGAGGAG-3 3 - GGTCTCTATATAGGTCTCTCCTC-5 5 -GAGGATGATGAGGATGAAGAAGAGATCGAGGTGAGGTCCAGAGATATATCCAGAGAGGAG-3 3 -CTCCTACTACTCCTACTTCTTCTCTAGCTCCACTCCAGGTCTCTATATAGGTCTCTCCTC-5 GluAspAspGluAspGluGluGluIleGluValArgSerArgAspIleSerArgGluGlu E D D E D E E E I E V R S R D I S R E E
Gibson Assembly Cloning Gibson assembly
Gibson Assembly Cloning Gibson assembly
Gibson Assembly Cloning A three component, one-pot reaction mixture containing: T5 Exonuclease Phusion polymerase Taq DNA ligase True in vitro ligation that is multi-fragment compatible DNA homology typically requires 30bp overlap Sensitive to termini that form stable single-stranded DNA structure
Golden Gate Cloning
Custom TALEN gene synthesis
Custom TALEN gene synthesis cont d
Red αβγ Cloning Redαβγ Cloning also termed λ- mediated recombination Requires E. coli strains which express the phage derived protein pairs Redα/Redβ from λ phage Protein pairs are functionally similar Redα (5-3 exonuclease) Redβ (DNA annealing proteins) Interaction required to catalyse homologous recombination Red γ is required to inhibits the host RecBCD exonuclease V
RED recombinase gene disruption in E. coli One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products Kirill A. Datsenko, 6640 6645, doi: 10.1073/pnas.120163297