Future Work: Sequential addition

One of the future expansions of the BRIDGE protocol that we have discussed involves using it to directly introduce genes in the genome next to each other without using the BioBrick method before-hand. For example, if you wanted to insert four genes with the steps described in the protocol section, it would take eight steps. If you do this with the method shown in Figure 1 below, it would only take four steps.

Figure 1: Using BRIDGE to directly insert genes into the genome.

At the first step of the process, the first antibiotic resistance gene 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, although this would only be to confirm the persistence of the marker).

The final construct would look as shown in Figure 2.

Figure 2: The theoretical final construct after using BRIDGE to directly insert genes into the genome.

The steps above are purely theoretical and have not yet been tested, but the principle behind them is not too distant from the original method, so it would be nice to attempt it if anyone ever gets the chance.

Future Work: A working protocol

So far we have been unable to produce cat/sacB recombinants using the protocol we have written up on this wiki. We believe we know where the problems lie.

We initially suspected that the plasmid containing the recombinase genes was incorrect, however a restriction digest of this with EcoRI gave the bands expected for that plasmid (please see the relevant lab notes).

We then thought that perhaps the cat gene was not very chloramphenicol resistant, but we have demonstrated its growth on cml40 and the titre indicated no chloramphenicol resistance at all in our transformants (see BRIDGE protocol and characterisation).

We eventually discovered notes on the protocol indicating that JM109 and DH5alpha are not suitable hosts for lambda recombination. Knowing this we have switched strain to K12. We are also now using kanamycin resistance instead of chloramphenicol resistance.

If these experiments still don't work then the induction step needs to be change. For example, L-arabinose is essential but it might be needed in higher concentrations. We hope to be able to give further updates on this matter at a later date.

Future Applications

We are hoping to submit the BRIDGE protocol as a new RFC (request for comments) to the Registry when we can confirm that it works.

Our own goal with this would have been to use it to insert multiple light sensors into a strain of E. coli by replacing the endogenous repressors that they use in their readout system. For example, we could replace the endogenous trpR with the LovTAP sensor and readout system BioBrick (BBa_K322999). This would remove all background noise from trpR at the same time as adding the light sensor.

This sort of protocol could have uses in areas of research requiring the addition of multiple genes to an existing genome. The advantages of the BRIDGE protocol over more traditional methods of BioBrick insertion have already been documented here. For example, there are Ph.D. students working in our lab working on butanol resistance and cellulase production in E. coli and Citrobacter. Both of these attributes involve multiple genes and have so far been transferred to their hosts in plasmids using normal BioBricking method. With BRIDGE these could be inserted into the genome quickly and efficiently, requiring no lasting selection markers.

Some studies use a controllable promoter to determine appropriate expression levels. Once you have determined this level of expression and want to have a consistent output without continuously adding the activating or inhibiting factor, you can use the BRIDGE protocol to replace just the promoter with one that has the appropriate expression output without repeating the entire construct and transformation.

Throughout this wiki there are words in bold that indicate a relevance to human aspects. It will become obvious that human aspects are a part of almost everything in iGEM.