Team:USP-Brazil/ConceptualFramework/Elimination
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Pathogen Elimination
For a successful paratransgenesis project, you will surely need lots of things - but the most important: it has to work! Picking the right effector is a key part of the design, specially since it may help determine what kind of genetic circuit you need to create.
Do you need a fast response or a sensitive one? Or both? Is your effector a single protein, or do you need a metabolic pathway to synthesize it? Does it only kill the targeted infectious agent, or does it affect your chassis as well? There are many questions to be answered when picking a method of elimination for your circuit, but we promise it’s not as complicated as it looks at first! Let’s go by parts:
Killing vs. Stopping Growth
When designing a circuit it’s important to determine what exactly do you want the output to be. Killing the pathogen may seem like the no-brainer choice here, but it isn’t always as simple as it sounds. Sometimes, the effector may work really well against the pathogen - so well, in fact, that it may be toxic to the host bacteria (or even the mosquito!), like the Scorpine protein we clonned for our project (Wanna check out more? Read the Results page here and scorpine’s Registry page here). Sometimes, an effector that kills the pathogen may be too complicated to produce in your chassis, requiring a long synthesis pathway. In the end of the day, it really depends on what kinda related literature you can find about it - and we compiled a few of our favorite ones at our Collection of Parts page, just in case they might come in handy for someone.
Nonetheless, you can still develop a paratransgenesis tool: use something that stops the pathogen from growing. That’s what we did with EPIP4, for example, a peptide that stops the malaria parasite from penetrating the mosquito’s epithelium. Doing a bit of a background check on your pathogen, you can probably find development stages that are an easy target for your inhibitor. Or if it’s something like a virus, just stop it from replicating altogether!
Fast vs. Sensitive
Another thing to consider, is how fast you need your circuit to be producing your effector. After all, no point in stopping the pathogen’s replication/growth if your system can only do it after it’s too late! If you’re curious about just how much does speed factor into this, check out our Modelling page where we talk more about that!
Sometimes, increasing a system’s speed may mess up a little bit with its sensitivity. But sensitivity is also an important thing to have in mind, specially if your detector depends on it! If it does (like ours did) make sure to check out the Detection page.
My effector is perfect, but it also kills my chassis! Do I have a problem?? Help!!
Not at all! Or at least not that much. Although it is a bummer when that happens, that is far from bringing your paratransgenesis project to an early end. First of all, it depends on the concentration. Since your GMO is probably gonna outnumber the pathogen, your effector may kill all of the pathogen and most of your chassis, leaving just enough to replicate and continue inside the insect vector.
Secondly, that is one of the benefits of a rational approach including a detector. This reduces the fitness cost for your host chassis, making it less damaging for your chassis on the long run (and for non related microorganisms, of course)!
My effector can only work outside the bacteria... how will it reach the pathogen??
Easy, using secretion systems! Bacteria have a great variety of secretion systems, and some can even be synthetically expressed on other hosts. The best approach is to look for endogenous secretions systems and add a tag to your effector. But if that is not possible, you can try the E.coli HlyA secretion system, as we did. Doing all of this, your effector will be good to go!
