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The aim of this module is to provide the iGEM community with the tools we used to delete genes in Escherichia coli. The deletions were performed using the phage λ Red system, as described by Datsenko and Wanner[1]. The toolbox is composed of two plasmid helpers and two antibiotic resistance cassettes BioBricks, adapted to iGEM standards. Basically, one could delete a gene with only this toolbox and a couple of primers designed specifically for the target gene.
We think that a simple way to delete genes could be a great asset in the context of designing new biological systems, and thus could be useful to future iGEM participants. Provided that the gene is not essential to the microorganism, the deletion could be constructed for different purposes, and open up new approaches in iGEM's synthetic biology projects, together with the BioBricks system. For example, removing a specific gene could enhance a particular metabolic pathway, like we did for the hydrogen production. Moreover, it could be easier to delete a gene hindering the production of a desired product than to over-express enhancer genes. It could also be useful if a gene is interfering with the function of a BioBrick.

 

How it works

 

In order to delete a gene, the first step is to perform a PCR using specific primers and a resistance cassette as template. The fragment is used by the Red system to replace the target gene with the resistance cassette, and the recombinant clones are then selected on an appropriate selection medium. The antibiotic resistance gene is then removed, so as to allow several deletions in the same strain while avoiding multiple resistances. Before explaining in details those steps, we will first introduce the different elements.
The first plasmid helper (PH λ) contains the three genes of the λ Red system: γ, β, and exo, necessary for homologous recombination. The γ gene encodes Gam, which prevents an exonuclease of E. coli from inhibiting the homologous recombination, while Exo is a 3’-5’ exonuclease preparing the PCR product for the homologous recombination reaction. The product of β, Bet, is the recombination enzyme.
The second plasmid helper (PHFlp) contains the flp gene encoding the Flippase, a site-specific recombination enzyme (Flp). Flp recognizes small sequences called Flippase recognition target (frt), and, in a way similar to the Cre-Lox system, catalyzes a site-specific recombination reaction between the two frt sites. In this case, it is used to remove the frt-flanked antibiotic resistance gene that replaces the deletion target gene.
Both plasmid helpers are low copy thermosensitive replicons, which means the plasmids do not replicate at 42°C. Thus, the plasmids are easily cured after the different recombination steps and the strain deleted of the target gene do not contain neither plasmids nor antibiotic resistance gene. Moreover, expression of the different genes is conditionally induced at 42°C which provides a strict control of activity.

The antibiotic resistance cassettes plasmids (RCP) are plasmids with a resistance gene flanked by two frt sequences (fig.2). There are two resistance cassette plasmids, one with a kanamycin resistance marker (RCPKan) and the other with a chloramphenicol resistance marker (RCPCm)

The primers used for the homologous recombination step contain a constant 3’region and a variable 5’ region (fig.3).  The sequence of the 5’ variable region depends on the target gene and is homologous to the sequence flanking the target gene.   The 3’ constant region is the priming sequence for the resistance gene PCR amplification.

With these elements, a gene deletion can be constructed in three main steps (fig.4).

Step 1: PCR

The first step consists of a PCR to produce the sequence which will replace the target gene. Using the frt-flanked antibiotic resistance gene carried by the RCP as template together with the primers designed specifically for the target gene, the PCR product is a sequence which can be inserted into the E. coli chromosome by homologous recombination with the Red system (fig.5).