BBSRC MIBTP Site directed mutagenesis, reverse genetics and complementation

1. BBSRC MIBTPSite directed mutagenesis, reverse genetics and complementation Chris Thomas, Joanne Hothersall, Yusra Al-Sammarraie, MukulYadav Biosciences, University of Birmingham

2. Mutational analysis • Identification of gene responsible for a particular inherited trait • Identification of inherited trait associated with a particular gene • Identification of amino acids/nucleotides responsible for a particular property (promoter, operator, active site) • Determination of interactions between genes

3. Types of mutation • Point mutations/base substitutions • Deletions • Insertions • Frameshifts • Inversions • Translocations

4. Why reverse genetics? • Traditional genetics • Mutant phenotype > Identification of a Gene • Reverse Genetics • Identification of a Gene > Mutant phenotype

5. Complementation • Complete or partial restoration of a mutant phenotype to WT by provision of the WT gene elsewhere in the cell • Important to show that the defect observed is due to the gene identified rather than a defect in a different gene • Important to show that the defect observed is not due to a polar effect on down stream genes • Distinguish how many cistrons responsible for a particular phenotype

6. Making the mutation • Synthesise a mutant gene • Overlap extension and PCR

7. Making the mutation – quick change

8. Knowing it’s there PCR and sequencing PCR and restriction digest WT Mutant Insertion of restriction site creates a Restriction Fragment Length Polymorphism

9. Putting it back into the genome

10. K A Datsenko and B L Wanner (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences 97, 6640–6645.

11. Datsenko and Wanner (2000) • Lambda Red recombinase – needs >35 nt arms • Linear fragment • AbR gene flanked by FLP recombinase targets Gene X

12. Datsenko and Wanner (2000) • Advantages • Short synthetic arms • Selectable mutation • AbR easily deleted by FLP and Frt • Disadvantages • Requires linear DNA fragment to enter bacteria • Leaves a scar at the site of mutation • Secondary alterations in the chromosome due to lambda Red system

13. Gene Doctoring • As Datsenko & Wanner but • DNA fragment generated in vivo by IsceI meganuclease • sacB gene used to select bacteria that have lost the donor plasmid • Lee DJ, Bingle LEH, Heurlier K, Pallen MJ, Penn CW, Busby SJW and Jon L Hobman JL (2009) Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains. BMC Microbiology 9:252

14. DNA fragment generated in vivo by IsceImeganuclease AbR sacB ISceI x x

15. Forced integration and excision by homologous recombination • Mutant cloned in suicide plasmid with sacB • Transfer to recipient bacteria – selection isolates products of integration event • Purify integrants very carefully and check for sucrose sensitivity • Remove selection and allow excision • Isolate products of excision because they become resistant to sucrose

16. Suicide vector requires integration to establish AbR AbR

17. Suicide vector for non-enteric bacteria

18. Counter-selection • sacB encodes levan sucrase • Sucrase is extracellular in Gram +ve bacteria • Levan accumulates in periplasm of Gram –ves • This inhibits growth glucose-fructose glucose-fructose-[glucose-fructose]n

19. Other vectors for forcing integration • R6K oriV plasmids replicating in host providing Rep protein from the chromosome oriV rep

20. Other vectors for forcing integration • Non-mobilizable plasmid replicating in bacteria with conjugative plasmid – select for transfer via cointegrate formation Rare recombination cointegrate

21. Construction of in-frame deletions OR Plasmid excision Mutant OR W.T. 500 bp 500 bp TARGET GENE FP1 FP2 RP1 RP2 PCR Construction of in-frame deletions

22. A. Kassem El-Sayed, Joanne Hothersall, Sian M. Cooper, Elton Stephens, Thomas J. Simpson, and Christopher M. Thomas (2003) Characterization of the Mupirocin Biosynthesis Gene Cluster from Pseudomonas fluorescens NCIMB 10586. Chemistry & Biology 10, 419–430. Hothersall, J., Wu, J., Rahman, A.S., Shields, J.A, Haddock, J., Johnson, N., Cooper, S.M., Stephens, E., Cox, R.J., Crosby, J., Willis, C.L.,Simpson, T.J.and Thomas, C.M. (2007). Mutational analysis reveals that all tailoring region genes are required for production of polyketide antibiotic mupirocin byPseudomonas fluorescens: pseudomonic acid B biosynthesis precedes pseudomonic acid A. J BiolChem282, 15451-15461.

23. Design Exercise I • Aim: to design primers to change one amino acid into another and create a restriction site change that can be used to detect the change in PCR products.

24. Designing the mutation • Overlap extension and PCR

25. Design Exercise I • You have been allocated one gene from the tailoring functions of the mupirocin biosynthetic cluster • Identify your assigned gene and copy the DNA sequence for the open reading frame plus at least 500 bp upstream and downstream

26. Design Exercise I • Go to the protein sequence of the gene product at NCBI • Perform a blast search to find related proteins • Identify one or more highly conserved amino acids that may be essential for function of this and related proteins • Choose one amino acid that you will plan to mutate

27. Design Exercise I • Identify the DNA sequence corresponding to the amino acid that you have chosen. Change the triplet so that it will become an alanine or a different amino acid if you feel that you can argue for something else. • Examine the sequence overlapping the change to see if it causes a change in the restriction pattern. • If not then see if you can design further silent changes in the sequence so the restriction pattern does change.