Sunday, 23 April 2017

A note on Mutazyme and Manganese

Mutazyme is great as it is not as biased as manganese mutagenesis which requires imbalanced nucleotides to correct, but annoyingly cannot reach a really high mutation rate. The maximum number of doublings is lower than the number of PCR cycles, so that imposes a limit to about 20 doublings. Assuming an error-rate of 0.9/kb/doubling, the hard limit is about 20 mutations per kb, which means that a 100 AA protein cannot be mutated to more than an average of 7 nucleotide mutations, which means 4 amino acid changes* (70% variants have between 2–5 mutations).
So what happens when manganese is used to increase the error-rate?
Curiously, the Mutazyme PCR reaction fails if 1+ mM manganese chloride is added. It gives a faint band, not multi-banding. Mutazyme is simply a homologue of Pyrococcus furiosus polymerase that lacks the proof-reading domain. The dNTP concentration is the same as normal PCR and the primers only twice higher. The odd thing is that there is a secret stabilizing protein with it. So most likely this sensitive enzyme cannot cope with manganese.
This is not really the end of the world as the mutation rate hoped for is too high for epPCR (redundancy issues and frameshifts), while degenerate primer based strategies are more recommended, but it's a shame it does not stack.


*) 58% of nucleotide mutations are amino acid mutations if all mutations are equal. This is an empirically determined figure.

Tuesday, 11 April 2017

A simple hack for a phylogenetic Noah's ark dilemma

Ever had an endless list of bacterial names that needed a trim?
Ever see a tree where the bacterium chosen is not the famous one, but it's cousin? Or actually a tree where you don't recognise a single name?
The issue of picking bacteria from a list is what I call Noah's ark dilemma. This term is used generally for the biblical problem of the size of the boat required for all the animals in existence (except dinosaurs). Here I mean it picking the most meaningful bacteria from a list. In the past year, I have come to rely on a simple solution: Pubmed popularity.

Sunday, 26 February 2017

Peak height variation

Sequencing a plasmid pool containing a sequence with a randomised codon can reveal the frequencies of the bases are (Acevedo-Rocha et al., 2015 ).
The problem is that sequence traces are not consistent. Some peaks are bigger than others and beyond a certain point the traces get messy. So how does that affect the prediction of the base frequencies?

Friday, 6 January 2017

Top 5 useful facts about the MDS42 strain

MDS42, also known as the Blattner strain, is a strain made in 2006 by deleting 13% of the genome of E. coli K-12 MG1655. Unfortunately, as tools go, it rivals IKEA in cryptic instruction manuals.

Wednesday, 30 November 2016

Saturday, 12 November 2016

The heteroduplicity of error prone PCR plasmids

A mix of wt and
mutant...
In an error prone PCR the ep-aDNA is ligated onto a plasmid backbone and transformed. When assessing the diversity from a naïve plasmid pool, something odd is seen: some bases are mutated but not to saturation. This is often just dismissed or simply overlooked, but I suspect it is actually something interesting...

Thursday, 10 November 2016

DNA gyrase for better yields

Transformation efficiency is a key part of library making... Which gets a bit tricky with large plasmids. A forgotten 90s paper would appear to have a solution, if it were not for a catch.