Molecular Biology Techniques Supporting IBBE

Transcription

1 Molecular Biology Techniques Supporting IBBE Jared Cartwright Protein Production Lab Head Contact Details: Phone

2 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

3 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

4 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

5 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

6 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

7 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

8 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-->

9 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

10 PCR Polymerase Chain Reaction (PCR) (Saiki et al. 1988) ds DNA containing target sequence Add primers and Taq Polymerase Multiple copies

11 Purified PCR Product 5 CTCAGGATTCATGTTTAAGGTAATCACTGTGTGCTTATG 3 GAGTCCTAGGTACAAATTCCATTAGTGACACACGAATAC GCTATTCGCCTTCAACAGTCTTAATGTGTCGTCCGAGTATGTTTATGAGGG CGATAAGCGGAAGTTGTCAGAATTACACAGCAGGCTCATACAAATACTCCC TTTGAAG AAACTTC TACTTACAACCTCAAGAATGAACGAAGATGCATAAAAGCTTGCGC 3 --ATGAATGTTGGAGTTCTTACTTGCTTCTACGTATTTTCGAACGCG 5

12 Digested PCR Product BamHI GATTCATGTTTAAGGTAATCACTGTGTGCTTATG GTACAAATTCCATTAGTGACACACGAATAC GCTATTCGCCTTCAACAGTCTTAATGTGTCGTCCGAGTATGTTTATGAGGG CGATAAGCGGAAGTTGTCAGAATTACACAGCAGGCTCATACAAATACTCCC TTTGAAG AAACTTC TACTTACAACCTCAAGAATGAACGAAGATGCATAAA --ATGAATGTTGGAGTTCTTACTTGCTTCTACGTATTTTCGA HindIII

13 Recombinant Vector Product Vector DNA Ligase -GATTCATGTTTAAGGTAATCACTGTGTGCTTATG -GTACAAATTCCATTAGTGACACACGAATAC GCTATTCGCCTTCAACAGTCTTAATGTGTCGTCCGAGTATGTTTATGAGGG CGATAAGCGGAAGTTGTCAGAATTACACAGCAGGCTCATACAAATACTCCC TTTGAAG AAACTTC TACTTACAACCTCAAGAATGAACGAAGATGCATAAA- --ATGAATGTTGGAGTTCTTACTTGCTTCTACGTATTTTCGA- Ligase Vector DNA

14 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

15 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

16 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

17 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

18 In-Fusion cloning

19 In-Fusion cloning Other uses Multiple fragment cloning

20 In-Fusion cloning Other uses Site-directed mutagenesis

21 In-Fusion cloning SDM cont d

22 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

23 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 CTCCTACTACTCCTACTTCTTCTCTAGCTCCACTCCA-5 5 -AGATCGAGGTGAGGTCCAGAGATATATCCAGAGAGGAG-3 3 -TCTAGCTCCACTCCAGGTCTCTATATAGGTCTCTCCTC-5 InFusion 5 -AGATCGAGGTGAGGTCCAGAGATATATCCAGAGAGGAG 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

24 Gibson Assembly Cloning Gibson assembly

25 Gibson Assembly Cloning Gibson assembly

26 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

27 Golden Gate Cloning

28 Custom TALEN gene synthesis

29 Custom TALEN gene synthesis cont d

30 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

31 RED recombinase gene disruption in E. coli One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products Kirill A. Datsenko, , doi: /pnas