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Team:Heidelberg/Human Practice/Phips the Phage/Project Sensing
From 2008.igem.org
Phage project
The phage project is concerned with engineering a module, which is able to kill pathogenic bacteria. These can be species, which invade wounds, for example, and lead to an infection, which can, in the worst case, end with the death of the patient due to a septic inflammation. There are also other bacterial infections inside the human body like pneumonia, for which our method could be a possible treatment.
Since bacteriophages are very efficient natural devices to destroy bacterial populations (as described in the virus section), we decided to use the power nature already invented for their project. But why is it reasonable to invent something new to kill bacteria, since we already have very powerful tools to do this: antibiotics? Well, you may have heard on the news, that there are many pathogenic bacterial strains that have become resistant to antibiotics, like the one that causes pneumonia. The diseases they cause cannot be cured with antibiotics any more.
The general idea is to bring a bacteriophage into a pathogenic bacterial cell. There it will be able to carry out its intrinsic killing mechanism to destroy this cell. A very important thing to our team was that the phages are specifically transferred to pathogen cell – because otherwise one could just take bacteriophages and apply them on the site of bacterial infection. This feature of targeting will be carried out by the bacteria the ‘sensing group’ of our team engineered.
The tasks the phage group had to carry out were to engineer the bacteriophages in a way that it could be stably packed into the ‘killer bacterium’ (the one that will kill the pathogen) without killing this useful cell and find a method to transfer the phage into the pathogenic cell, where it should get active and kill the pathogen.
We will go through these steps in a very general way to see, how the engineering work has been done and how this system works. For detailed information see the project description of this group.
To change the properties of the lambda phage – to make it stay stably in the ‘killer’ cells for example, the genome of the phage has to be changed. This is because the genome codes for all the proteins of the phage and these are responsible for the shape, the replication and the lysis of the host cells. To change the properties of these proteins, the underlying genes have to be changed. This can be done by genetic engineering using standard molecular cloning techniques (link).
What we wanted to get rid of specifically were the genes which encoded for the lysogenic property of the phage (see section viruses). Since we want to engineer a very efficient killing device, it would not be very reasonable to have a bacteriophage, which is able to integrate into the pathogen’s genome and stay there without killing it.
The properties we wanted to add to the phage were one to be able to transfer it to the pathogen and one to make it fluorescent to visualize it.
Generally, the genome of the lambda phage can be handled like a standard plasmid vector. But working with the genome of the lambda phage is far from being trivial, even to experienced scientists. The difference to the common lab work is that the lambda genome is huge compared to ordinary plasmids, genetic engineers work with: It is 10 times as big as many standard plasmids. And there is another big problem: Since the lambda genome is not an optimized tool for genetic lab work, it contains few useable restriction sites. Remember, these are the sites, where you can cut a DNA-strand – to put some new base pairs (and thereby information) into the sequence or to cut some out. But you can only modify such a huge DNA in the given time we had by molecular cloning. Therefore we had to cut out much bigger fragments than we wanted to, which still contained genes we needed, because we wanted to discard some genes from the genome, and therefore we had to use the few restriction sites available, even if they didn’t fit our purposes very well. The genes we unwontedly cut out had to be put into the phage genome again together with the other genes we wanted to add, to give the phage new properties.
How can the lambda phage bee detained of lysing the killer bacteria?
There is a protein called the lambda repressor or cI, which completely represses the activity of the lambda phage in a bacterial cell. This protein is naturally made by the lambda phage under certain conditions. It binds to the genome of the phage and prevents the transcription of the genes necessary for the induction of an infection cycle: It inhibits the RNA-polymerase from binding to the lambda DNA. No proteins can be made, which means that the phage cannot carry out any activity (see section Genes and Proteins). But why does the lambda phage make such a protein naturally, since it can repress its own activity?
Well, the lambda phage uses it to repress other lambda phages. If one phage has infected a bacterial host and carries out a lysogenic cycle, it produces this protein. Since it is not active in this mode anyway, it does not matter for itself. But it is important, if other bacteriophages infect this particular host cell. Their activity is repressed from the beginning on and they neither can carry out lytic nor lysogenic cycle. Therefore their DNA will just be degraded by the host cell. So the original invader defends and protects its host cell against other lambda phages with the help of the lambda repressor protein.
If we now bring this protein into our killer cells, the lambda phage we will later put in will be inactive and can not kill our engineered cells.
How can we make the phage fluorescent?
Well, to be precise, we do not make the phage fluorescent, but the phage will give this property to all cells where it is in. This property is gained by using fluorescent proteins. They are able to absorb light and emit light of lower energy and thereby glow. One protein with this feature is taken from a jellyfish and called Green Fluorescent Protein (GFP). It is the oldest and most broadly used fluorescent protein in biotechnology. Because it had such a great impact on this science and improved it a lot, this year’s Nobel Prize in chemistry was awarded for the discovery of GFP and its applications. If GFP is made by a cell and you illuminate this cell with UV-light, you can see a green light coming from this cell. In adding the gene for GFP to the genome of the lambda phage, the cell hosting this phage is able to produce GFP and can thereby be detected.
How can we make the phage transferable to the pathogen?
Generally, we do not want to transfer the phage in its extracellular form to the pathogen, in which the DNA is enclosed in the protein coat and the bacteriophage is able to infect the pathogen in its own. Because to set this form free, the host cell of the phage has to be lysed. We do not intend to destroy our killer cells to set the phage free, because then they could only be active for a short period of time. In finding a mechanism to transfer the phage without the lysis of the host cell, the killer bacteria could be active over a longer period of time and therefore really kill all the pathogenic bacteria
So the idea we had was not to transfer the whole phage, but only its genome, since this has all the properties needed to lyse the target cell. A very powerful natural tool to transfer genetic information is bacterial conjugation (link). So the thing to do was to make the lambda genome accessible for conjugation. Normally, as I explained in the conjugation section, there are conjugative plasmids, which carry all the genes needed for their own conjugation. Since these are quite a lot of genes, we did not want to put them all into the lambda genome. But we found an easier solution: Binary vector systems for conjugation.
Most of the proteins, which are essential for conjugation, do not have to be on the plasmid that is transferred. This makes sense if one imagines that the proteins are produced in the bacterial cell and can spread there and get to the working places. So it doesn’t matter if a conjugative protein is produced from the chromosome, from an additional plasmid or from the plasmid which will be transferred. So what we had to do was to take a conjugative plasmid, which produces all the essential conjugative genes and put it in the killer cells?
But how do these conjugative proteins know which plasmid they should transfer? For this task, there exists a sequence in the plasmid to be transferred, which is called oriT. It tells the conjugative proteins: ‘Hey guys, this is the plasmid you are looking for!’ and provides a site for them to bind to and to get active. So in adding this oriT site to the lambda genome, the phage DNA can be transferred to other bacterial cells.
Summary
And remember the reason why it is stable in the host cell and not lysing it? Because of the expression of the lambda repressor protein! But this protein is not expressed in the target pathogen cell. So once transferred there, the phage DNA can be transcribed, and the proteins for the lytic cycle can be made. There is no possibility to carry out the lysogenic cycle, because we removed the genes necessary for this mode from the genome. So the lambda phage will carry out its work in the pathogen and destroy it.
And now comes the best part of this system: As I already told you, phages are multiplying during the lytic cycle. So when the pathogen cell is destroyed, approximately one hundred new phages are release (originating from one infecting phage). So the number of phages is multiplying with every round of infection: We are having a Domino Effect. This clearly contributes to the aim to kill the whole population of pathogenic bacteria in the inflammatory tissue.
