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, Mukul Yadav
• 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 gt Identification of a Gene
• Reverse Genetics
• Identification of a Gene gt 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 its 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, 66406645.

11
Datsenko and Wanner (2000)

• Lambda Red recombinase needs gt35 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 9252

14
DNA fragment generated in vivo by IsceI
meganuclease
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-fructosen
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
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, 419430. 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 by
Pseudomonas fluorescens pseudomonic acid B
biosynthesis precedes pseudomonic acid A. J Biol
Chem 282, 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.