Temporary mutator strains can be built by over-expressing a mutator allele such as mutD5 (a dominant negative version of mutD) which limits the cell’s ability to repair DNA lesions. The drawback with this method is that the strain becomes progressively sick as it accumulates more and more mutations in it’s own genome so several steps of growth, plasmid isolation, transformation and re-growth are normally required to obtain a meaningful library. One advantage of mutator strains is that a wide variety of mutations can be incorporated including substitutions, deletions and frame-shifts. As a result each copy of the plasmid replicated in this strain has the potential to be different from the wild-type. XL1-red is an E.coli strain whose deficiency in three of the primary DNA repair pathways ( mutS, mutD and mutT) causes it to make errors during replicate of it’s DNA, including the cloned plasmid. In this approach the wild-type sequence is cloned into a plasmid and transformed into a mutator strain, such as Stratagene’s XL1-Red. This eliminates the ligation step that limits library size in conventional error-prone PCR but of course the amplification of the whole plasmid is less efficient than amplifying the coding sequence alone. This is a variant of error-prone PCR in which wild-type sequence is first cloned into a plasmid, then the whole plasmid is amplified under error-prone conditions. There are a number of commercial error-prone PCR kits available, including those from Stratagene and Clontech. Although point mutations are the most common types of mutation in error prone PCR, deletions and frameshift mutations are also possible. ![]() The drawback of this approach is that size of the library is limited by the efficiency of the cloning step. After amplification, the library of mutant coding sequences must be cloned into a suitable plasmid. Here is a good review of error prone PCR techiques and theory. The PCR can be made error-prone in various ways including increasing the MgCl2 in the reaction, adding MnCl 2 or using unequal concentrations of each nucleotide. This approach uses a “sloppy” version of PCR, in which the polymerase has a fairly high error rate (up to 2%), to amplify the wild-type sequence. 8 Different Approaches to Random Mutagenesis 1. There are many ways to create random mutant libraries, each with it’s own pros and cons. Creating a random mutant library that contains enough variants to give you a good chance of obtaining the altered enzyme you desire is a challenge in itself. Sound easy? Well, of course it’s not that easy. Using a high throughput screen for GPCR activity (see here for examples) you could pick out the variants from the library that were temperature-sensitive or were activated by different ligands. Next, you could transform the library into a strain where the receptor would be expressed and apply a high throughput screen to pick out variants in the library that have the properties you are looking for. ![]() Each version (or “variant”) of the gene in the library would contain different mutations and so encode receptors with slightly altered amino acid sequences giving them slightly different enzymatic properties than the wild-type. ![]() How could you do this?įirstly, you would clone the gene encoding the receptor, then randomly introduce mutations into the gene sequence to create a “library” containing thousands of versions of the gene. Imagine, for example, you were studying a G-protein coupled receptor (GPCR) and wanted to create a temperature-sensitive version of the receptor or one that was activated by a different ligand than the wild-type. Random mutagenesis is an incredibly powerful tool for altering the properties of enzymes.
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