Team:ETH Zurich/Modeling/Genome Static Analysis

Restriction Enzyme Analysis

This section presents the computational investigation we performed in order to understand which restriction enzymes are optimal when used in our minimal genome approach, in order to find out which restriction enzymes cut the genome in fragments that most probably will lead to find the minimal genome in our reduction approach. Important is to note that this is a "statical" analysis, meaning that we do not include in the evaluation of the restriction enzyme optimality any prevision regarding to the effects its cutting patterns can have on cell physiology or cell system behaviour. We addressed questions regarding the cell system response after genome reduction using more advanced modelling techniques (a genome scale model) in the Genome Scale Analysis section. We focus here only on the insights that can be obtained using three kinds of "statical" information:

• the genome sequence of our strain of interest (E.Coli K12 MG1655).
• the annotation information of our strain of interest (E.Coli K12 MG1655).
• the recognition site patterns of each of the restriction enzymes we test.

Using computational tools and the above mentioned information we are interested in asking (and answering) the following questions:

• Which are the available restriction enzymes, their recognition sites and the fragments they generate after digestion?
• How is the distribution of the genes in each fragment related to the frequence of cutting?
• Is it possible to identify restriction enzymes that optimizes the probability of cutting out fragments of the genome but still keeping the cell alive (or better, do exist restriction enzymes that rearely targets fragment containing essential genes)?

Available restricion enzymes and digestion simulation

As source for the restriction enzyme to consider, we used the [http://rebase.neb.com/rebase/rebase.html REBASE database]. We found 713 restriction enzymes that spawn from 4 up to 13 cutters, some with complete specific recognition sites and some with unspeficisity properties. Since some of the restriction enzymes present the same recognition site sequence, we grouped them together as a single entity to be tested (216 groups). We downloaded the genome and annotation information regarding E.Coli K12 MG1655 from GenBank® database. We then simulated the digestion of E.Coli chromosome sequentially for each group of restriction enzymes and performed statistical analysis on the fragment pattern obtained. The following pictures summarize the distribution of the available enzymes regarding to their frequency of cutting (number of fragments after digestion):

It is possible to see that there are available a huge number of restriction enzymes that difest the chromosome in few to medium number of fragments (up to 10000 fragments) and relatively fewer that generate a high number of fragments.