Week 7: Genome Engineering


In this class we discussed methods to identify, assemble and transplant a minimal genome. Mycoplasma genitalium has the smallest genome of any organism that can be grown in pure culture. It has a minimal metabolism and little genomic redundancy. Consequently, its genome is expected to be a close approximation to the minimal set of genes needed to sustain bacterial life.

Using global transposon mutagenesis, Prof. John Glass and his group isolated and characterized the genes of M.genitalium into three categories: essential, growth-impeding and non-essential. Toward achieving rapid and large scale genome modification directly in a target organism, they have also developed a new genome engineering strategy that uses a combination of bioinformatics aided design, large synthetic DNA and site-specific recombinases.

Homework Assignment

Useful file for homework here: EMycoplasma mycoides JCVI syn1.0 transposon bombardment results This file (see Fig. 1) lists all the genes in the Mycoplasma mycoides JCVI syn1.0 minimal genome version and ells you what transposon bombardment revealed about each gene, i.e. essential (e), not-essential (n), or whether a disruption of that gene causes impaired growth (i).

Fig. 1: Provided file with transposon bombardment results.

Assuming we have a target bacterium of which we want to identify the essential genes for a minimal genome:

  1. Find common (i.e. highly conserved) essential genes among the species for which we already have “minimized genomes”. Subsequently compare them with the genome of the target bacterium.

  2. Selecting the genes with conservation above a certain similarity threshold, assemble the putative minimal genome for target bacterium and transplant it. The threshold should be chosen such that it compensates for the GC content and species specific mutations.

  3. See if the bacteria can grow at all, and monitor growth curve. Adjust threshold based on growth rate. The strategy is to start with a very low threshold such that the growth curve is identical to the native bacteria and keep pushing it higher (i.e. selecting fewer genes) until growth is impeded.

As an extra step we proposed to compare the identified essential genes with synthetic minimal cell (SML) genes and find the differences. We believe that there are two sets of genes essential for life: The ones common to the minimal genome and a SMC, encoding the Tx-Tl machinery and the ones that are not found in SMCs, i.e. DNA replication and energy production genes.

Fig. 2: Two sets of genes important for a minimal living cell.


Searching later on the related literature, we found a paper titled Essence of life: essential genes of minimal genomes by John Glass's group, where they describe in depth several answers to the question of the homework. Our answer matches their first proposed method (see Fig. 3 below).

Fig. 3: Methods for identification of essential genes by Juhas et al. .