Random mutagenesis using a mutator strain
Random mutagenesis using a mutator strain
This protocol has been confirmed to work for the following organisms.
Mutator strains are useful for generating random mutations in your gene of interest. These mutants are then screened or selected for your phenotype of interest (e.g. screening for "white" mutants).
This protocol uses the commercially available XL1-Red mutator strain, which contains null mutations in the mutS, mutT and mutD alleles and is thus deficient in the mismatch repair system. The mutation rate in the XL1-Red strain is therefore ~5000x higher than it is in wild-type Escherichia coli.
A library of random mutants is generated in an E. coli mutator strain, pooled and transformed into ET12567/pUZ8002 (or another suitable strain) for intergeneric conjugation into your Streptomyces. The standard ET Conjugation protocol is then used to transfer the library of mutant vectors into your desired Streptomyces strain. A suitable genetic screen or selection is then used to identify mutant(s) of interest.
The number of exconjugants that must be screened/selected to obtain mutant(s) of interest will of course depend on the size of the gene you are mutating and the nature of your genetic screen/selection. However, having high-efficiency protocols for all transformation and conjugation steps is recommended, as this will ensure that you screen as large a library as possible.
- E. coli XL-1 Red competent cells (TetR; Agilent))[Note 1]
- E. coli ET12567/pUZ8002 (KmR, ChlR)[Note 2]
- Streptomyces strain of your choice
- Your chosen vector for mutagenesis
- Primers for sequencing your insert cloned into this vector
- Liquid LB medium
- Liquid SOC medium
- Antibiotic stock solutions (See the Antibiotic stocks and working concentrations page for recommended antibiotic concentrations.)
- Solid LB agar containing antibiotic to select for your vector of choice
- Solid LB agar containing antibiotic to select for your vector of choice, kanamycin, and (optional) chloramphenicol
- Miniprep kit
- Sterile loop/toothpicks, spreader, Eppendorf tubes, pipet tips, etc.
- Transform your vector into XL-1 Red competent cells (Agilent) following the manufacturer's protocol and plate on LB medium containing the appropriate antibiotic to select for your vector. Incubate plate(s) overnight at 37ºC.
- Using a sterile loop or toothpick, pick a large number of transformants (~200 or more colonies) and pool these into one liquid culture (in LB medium containing the appropriate antibiotic to select for your vector). Incubate this culture overnight with shaking (250 rpm) at 37ºC.
- After the overnight incubation, make a miniprep of your now-randomly-mutated vector. (If desired, you may also sub-culture the culture into fresh liquid LB medium containing the appropriate antibiotics to select for your vector, for a further round of mutagenesis.)[Note 3]
- Transform the miniprep containing a library of randomly mutated vector into theE. coli ET12567/pUZ8002 strain, and plate on selective medium containing kanamycin and the appropriate antibiotic to select for your vector. (Depending on the genetic selection/screen you are using downstream, you may also wish to transform ET12567/pUZ8002 with the unmutated vector as a control at this point.)
- Conjugate your vector into your Streptomyces strain of interest via the standard ET Conjugation protocol.
- Pick and restreak exconjugant(s) with your phenotype of interest.
- To identify the mutation(s) responsible for the phenotype, you can either re-isolate the vector following a plasmid rescue protocol, or directly amplify the vector insert by PCR and sequence the PCR product. (See High GC PCR for tips.)
- Because mutations may have arisen in extragenic sequences on the vector, you should re-clone the mutant allele into the original parental vector and introduce it into your Streptomyces strain to verify the phenotype.
- The XL1-Red strain is resistant to tetracycline, but this marker may be lost at a relatively high frequency due to frequent mutations arising in this strain.
- The E. coli strain ET12567 contains a chloramphenicol resistance gene integrated into the genome. This marker does not need to be selected for during routine culture of the strain.
- The XL-1 Red strain has a relatively high mutation rate due to the mutations in mutS, mutD, and mutT. However, you may still wish to perform multiple serial subcultures to allow for more than one round of mutagenesis. The strain begins to accumulate mutations and grow quite poorly after ~3 rounds of serial subculture.
Muteeb and Sen (2010). Random mutagenesis using a mutator strain. Methods Mol Biol. 2010;634:411-9. doi: 10.1007/978-1-60761-652-8_29.
Protocol adapted for ActinoBase by Morgan Anne Feeney from the University of Strathclyde.