In vivo shuffling via heteroduplex amplicons

Bluescreen transilluminator photo showing
different shades of green.

In a previous post I discuss the heteroduplicity of epPCR plasmids. Namely often colonies in a library will have bases that have two equal variants. The most likely cause being that the amplicons do not anneal perfect and that the transformed plasmids are actually heteroduplexes. These can either divide before being corrected or are corrected by mismatch repair.
I did a sneaky experiment to see if this could be used to make protocol to shuffle mutations between variants and got a positive result, but possibly not as effective as hoped.

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...

JW numbers in the Keio

Hirotada Mori is one of the top five names* in E. coli genomics as his group built the Keio and the ASKA collections. Yet the strains from the Keio and ASKA do not start with HM, but JW. Here is why...

Methodological sabotage of growth rates

Following the interest in a previous post about analysing growth curves in Matlab I would like to discuss issues in growth curves that can arise from the methodological/biological side of things. Fitting the data is perfect if the data is perfect, if not, looking at what is wrong by eye is warranted for future corrections.

Growth curves can be divided into phases (lag, exponential, stationary and death) and each has its pitfalls.