Team:Heidelberg/7 October 2008

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Revision as of 07:18, 8 October 2008

Project description lambda phage

Antibiotics are a very powerful way to cure bacterial infections till today, but their power withers. More and more bacterial strains become resistant against different antibiotics, due to the even greater powers of evolution. Bacteria also have a very effective way to share those resistances with each other, not limited by species boundaries: the transfer of plasmids by conjugation (which will be investigated in more detail in the second part of the project). Examples for resistance problems in different infectious diseases… Microfilms as a biological hazard in hospitals..?

There are some alternatives to antibiotics, which are currently researches: the usage of bacterial toxins, the usage of probiotic bacteria and the usage of bacteriophages as antibiotics. In our approach we are focusing on a combination of the two latter ones. We designed probiotic bacteria, which will kill the pathogens in vivo / in situ and therefore avoid the side effects of a systemic drug application. As killing device we take an engineered lambda phage.

The lambda phages are a class of temperent bacteriophages. They have a 48502 bp genome consisting of linear double stranded DNA that circularizes at the so called cos sites if injected into the host cell. As temperent phages they are able to undergo either a lytic or a lysogenic cycle. Undergoing a lysogenic cycle, the infecting phages integrates its genome into the host genome and stays there as a stable prophage. It will not lyse the cell and multiply without an extern trigger – for example if the host cell is stressed. If carrying out a lytic cycle, the lambda phage also injects its genome into the host cell. This will then be replicated, viral proteins will be produced, first for the formation of new phage particles, which are formed via self assembly, and second for the lysis of the host cell, upon which the newly produced phages are released. The lambda phage has a very complex regulatory network, which controls which cycle to carry out. The key role in this process is taken by a protein called cI. This binds to regulatory sequences, that stabilize a transcription of the genome which is essential for the lysogenic cycle. The lysogenic proteins include an integrase (int), which integrates the phage genome into the host genome. If this is not present, the phage genome stays in the cell in a plasmid like state. For the induction of the lytic cycle from a lysogenic one, a protein called excisase (xis) is essential. This one cuts the phage genome out of the host genome and thereby allows the replication and transcription of it. During the lysogenic cycle, cI is also expressed continuously. If another phage injects its genome into the host cell of a prophage, cI binds to regulatory sequences on this second genome and prevents any transcription, thereby making the host immune to secondary infections. Our work focussed on designing a non-lysogenic phage, because we want to produce an efficient killer module for the probiotic bacterium. In preventing the phage to undergo a lysogenic cycle, we assure that it will kill the target bacteria as fast as possible and without extern triggers. For this task we aimed to discard the int gene from the phage genome, so that it would be incapable of integrating into the host genome and being lytically inactive.

Cloning with the lambda phage genome is tricky, because it contains few single cutter restriction sites. Therefore we had to cut out one fragment containing the int, and also the xis and another gene, the gam. Xis and int can and shall be discarded, but the gam gene codes for a.. that prevents the overhanging single strains at the cos sites from being degraded by host enzymes. So we would need the Gam gene in our engineered phage. This is why we designed an insert for the phage to put in the remaining genome after cutting out the fragment containing xis, int and gam. This insert contains gam, and an antibiotic selection marker to be able to select those bacterial clones that contain the lambda genome. We also added a GFP gene for visualizing and measurement purposes and an OriT (see protocol 2). To be able to later modify this phage insert again, we added special restriction sites to only cut out the non-phage genes.

To avoid, that our probiotic bacteria, that would transfer the phage to the target pathogenic bacteria, would also be killed by the phage, we used the natural way to make them immune. Therefore we took the lambda cI gene and put it behind a constitutive promoter. The cI protein will then prevent the expression of the lambda genome in our probiotic bacteria and prevent them from being lysed.

Results

Cutting the lambda phage

As restriction enzymes we took XhoI and XbaI to cut out the xis and int – fragment. The resulting parts are the 9 kb fragment and the rest genome of 40 kb. (picture and exact data). Because the lambda DNA comes linear, there are different fragments for the rest genome if put on a gel: the two ‘arms’ of the vector (kb) and some hybridized complete rest (40 kb).

The new insert (take, if it gets finished, the second strategy, maybe even with the test results of the first one – just mention it)

The new insert consisting of the gam, a GFP, a chloramphenicol gene and an OriT was designed by PCR (each part on its own) and after this cloned together in pBluescript. (cloning strategy)

The parts in pBluescript are functional (for conjugation results see protocol 2).

Ligation into the lambda vector

The insert was ligated into the purified lambda vector. Then the ligation was transformed and cultivated for 2 to 3 days at 28°C. This was done because the lambda DNA we were using had a temperature sensitive cI. This is stable at temperatures under 37°C and thereby preventing lysis of the cells, and at more than 37°C it changes its conformation to an inactive state and the lytic cycle of the phage will be induced. Therefore we could amplify the ligated lambda genome at 28°C to later extract it via miniprep.

Infection tests

Infection tests with the lambda phage had to be carried out at 42°C, so cI would be inactive and could not prevent the lytic cycle.

Outlook

Up to now, our system is a proof of principle that the usage of phages as a killer module for probiotic bacteria works. For a later application, either the host range of the lambda phage has to be altered, to broaden the range of target pathogens. Another way would be to use other pahges with other host ranges within the same system. The problem with this approach is, that they have different regulatory mechanisms and therefore the engineering work would have to start new with each phage.

The most elegant way would be to broaden the host range of the lambda phage. Since phages identify their host via specific receptors on their tail fibers that bind to structures on the surface of their host one would have to modify or exchange these receptors. One approach could be to make chimeric receptors out of the lambda regulatory part and the identifying part of the receptor of another phage.

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