Team:Edinburgh/Project

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BRIDGE: The concept

BRIDGE stands for BioBrick Recombination In Direct Genomic Editing. It is a non-standard method of creating BioBricks using homologous recombination instead of restriction digestion to directly insert new genes into the genome, without leaving a marker behind.

The first step of BRIDGE requires the deletion of existing DNA (probably a non-coding piece or a non-essential gene) to introduce a construct of two genes; one an antibiotic resistance gene, the other sacB, which prevents the host from growing on sucrose. After the first step we can select for cells which have taken up the construct by growing them on the relevant antibiotic.

The second step involves swapping the construct for another piece of DNA (e.g. a BioBrick construct). After this we can select for those with the new gene by growing the cells on sucrose.

BRIDGE: The advantages

BRIDGE has a significant advantage over the current method of BioBrick insertion. For one, vector independent - whole PCR constructs can be inserted directly into the genome in two steps in under a week, compared to the lengthy process of vector digestion and ligation required with normal BioBricks.

The other major advantage is that it will not leave a lasting marker in the genome. With most BioBricks we have to leave a marker (antibiotic resistance, GFP, etc) in our constructs so that we can guarantee their presence. This becomes an issue, a) when you want to use the organism in an industrial or environmental capacity, and b) when you want to insert multiple constructs (there is only a limited number of markers out there). With this system, the markers are removed every time you insert a new gene, so they can be used again and again indefinitely. You could essentially replace the entire genome with new genes.

Our Project

Our BRIDGE construct will contain chloramphenicol resistance (cat) and sacB. Both it and the desired gene will be inserted by homologous recombination using the lambda red system. For this we will need up and down-stream sequences of genes which we wish to replace.

To prove the principle of BRIDGE we will remove a non-essential, constitutively expressed gene from the E. coli genome and replace it with a well known marker, such as GFP. We also have several genes from a past project idea which we could delete to increase fatty acid synthesis, and further genes we could introduce which will result in the production of long chain alkenes from the excess fatty acids. This is not useful for our current project but it is a nice way to demonstrate the effectiveness of BRIDGE.

Eventually, BRIDGE will be used to introduce whole light producer-sensor constructs (we hope).

Sequential Addition

One of the future expansions of BRIDGE which we have discussed is using it to directly introduce genes in the genome next to each other without using the BioBrick method before-hand. If you wanted to insert 4 genes with the steps described above, it would take 8 steps. If you do this with the method below it would take 4 steps.

At the first step of the process, the first antibiotic resistance and sacB are introduced alongside the first gene. The antibiotic resistance gene can then be replaced with the next gene and a second antibiotic resistance gene, thereby cycling the antibiotic resistance such that selection is different at each step. At the last step, both markers are removed and the final constructs can be selected for by growth on sucrose (growth on sucrose can also be used as a negative control at each stage).

The final construct would look as below: