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Design

Memo-Cell project : Design

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To integrate a DNA sequence into the chromosome in a stable manner, the Phage recombination systems seemed to be appropriate, as this can allow the integration of a whole plasmid into the bacterial chromosome, as soon as it carrie an attP recombination site. By expression the phage integrase, one can easily trigger the plasmid integration into the chromosome via the recombination between the plasmidic attP site and the chromosomal attB site. Please click here for a detailed explanation

When integrating into the chromosome a plasmid via phage recombination, the original chromosomal recombination is disrupted and hence prevent any other integration. To achieve a sequential integration into the chromosome, we had to find a way to recreate a functional recombination site after the first integration, so as to be able to add another piece of DNA.
We could not directly excised the whole DNA sequence that had just been integrated, as no DNA sequence of the plasmid would stay on the chromosome to count. Hence, we had to chose another mechanism.

To do this, we have chosen to engineer the Tn916 transposon.
The Tn916 is a mobile 18kb DNA sequence that is efficiently excised from the chromosome when its recombinase are expressed. The excision process which allows the creation of a circular Transposon, requires the enzymes to interact with their binding sites which are located at the extremities of the transposon: left arm (280bp) and right arm (180bp).
The integrase proper functioning also requires some specificity of the DNA sequence flanking the arms on the chromosome on around 20bp. During the excision process, the transposon ends are joined by the 6bp flanking either the left arm or the right arm, called the coupling sequences. After the excision of the transposon, only one of the the coupling sequences is the only leftover of the transposon that is left on the chromosome.

We severely mutated the flanking sequences of the transposon by replacing them with half of the phage recombination site so that once the transposon excised, a functional phage recombination would be regenerated on the chromosome. Because some nucleotides of the flanking sequences were essential for a functional excision of the transposon, we had to keep them and so, we could not just paste the original half recombination.
Hence, mutations have been inserted both in the flanking sequences and the phage recombination site.
Moreover, we had to drastically reduce the size of the Tn916 (18kb) as playing around with such big DNA sequences is experimentally not easy. We have then reduced its size of 90%, cloning between the left and right arms either a Kan-resistance cassette (~1kb) or a Kan-resistance and a Lac-Z alpha cassette (~1,8kb). The total size of our mutated transposon was either 1,4 kb or 2,2 kb.
The efficiency of excision using the reduced transposon with mutated arms has been assessed (protocol and detailed results in the result page).
We have also assessed the efficiency of recombination using the mutated phage recombination sites (protocol and detailed results in the result page).

Now that we had working systems to integrate a DNA sequence into the chromosome and another one to reform a functional recombination site, we had to integrate them together so that these two could work sequentially.

To have a sequential integration of DNA sequences one after the other on the chromosome, the new combination site after excision had to be adjacent to the DNA sequence that had just been inserted.

One could have thought to place the mutated Tn916 directly in the plasmid that will be integrated in the chromosome. But then, the transposon excision would happen not only on the chromosome but also in the leftover plasmids in the cytoplasm, drastically reducing the chance to have a plasmid still containing the Transposon at the next round of counting.

However, as excision of the transposon requires not only the presence of both the Left and the Right arm, but also that these two arms are on the same DNA molecule, we constructed our integrated plasmid with only half of the transposon, with the left arm only.
The right arm would be located on the chromosome after the phage recombination site