Hi, my name is Jason Whitfield and as part of a team here at EMBL for Synthetic Biology in Action project, I’ll be taking you through the genetic firewalls to horizontal gene transfer. In an effort to greater understand synthetic biology and its emerging contributions to society. So, synthetic biology is a field that is marrying engineering, chemistry, and biology, building on efforts of people who’ve come before us in biology through the creation of sequencing and the Genomics Project, to help us create new to nature functions whereby we can help microbes such as Pseudomonas putida create things such as biofuels. To help us lessen our reliance on fossil fuel methods that we currently use. Also to create therapeutic drugs to help us dispense with the messy and often time-consuming and expensive methods that organic chemistry has given us. Third, we can also create biosensors, whereby we can actually selectively look for a molecule of interest in a biological system or even in the environment. And lastly, or more importantly, we can in fact do bioremediation, whereby we can clean up some of the pollution that has accrued over man’s occupation. To do this, we need to be able to contain these genetically modified organisms that we’re creating. The Asilomar Conference of 1975 highlighted this, and since then a lot of genetic and physical containment strategies have been put into place. And these largely rely on the idea that we keep bacteria in a lab or in a reactor. However, bacteria can transfer their DNA. This is a process known as horizontal gene transfer. This is when DNA from one organism is transferred to another, conferring that new function to the recipient organism. However, DNA can survive in the environment for months in the right conditions. So even though an organism is dead, it’s DNA will still survive. And this in fact can be taken up also. This really highlights the idea that we need to create genetic barriers to this uptake or conferrence of genetically modified function. This really highlights the need for us to create a genetic control mechanism by which we can prevent the spread of this DNA. To do this, we need to go to our basic biology. And this involves looking at DNA replication, which is a high fidelity process involving the concerted and complex interplay of a variety of enzymes and proteins to create conventional DNA replication. And this however is not always true. And sometimes mistakes can occur whereby uracil can be misrecognized and therefore misincorporated into the DNA strand as thymine. And this occurs through a variety of mechanisms. Most often governed by the ratio pool of uracil to thymine. However, there are enzymes involved in the cell that can actually regulate this process. DUTP nucleotide hydrolase, and this one, what it does is actually converts UTP to the monophosphate variant, liberating pyrophosphate and doing so, shuttles it down the biosynthetic pathway towards thymine formation. Thus, lowering the pool and lowering its incorporation. Another process the cell has in its proofreading is UTP uracil DNA glycosylase. This enzyme moves along the DNA strand and flips out nucleotides, assessing their identities, and when it finds a uracil, it cleaves the backbone bonds, thus initiating the uracil repair response. So another mechanism that can also occur is the deamination of cytosine to the uracil, thus increasing the pool of uracil present in the cell and level of misincorporation. So, one of the kind of ethos of synthetic biology is to hijack these native functions and repurpose them. And so, the idea for this project is to hijack this misincorporation process as a means of creating a genetic control in the organism, Pseudomonas putida. To kind of create a new genetic language, whereby we have a genetic firewall that ensures that if a plasmid is present in an organism, it will be rich in uracil and its survival will be dependent on it staying in that target host. And conversely, creating an organism that has a brittle genome which will accumulate these uracils and should it come into contact through mating or other methods with the copies of DUT and UNG, it will in fact be degraded. To look at the first example of these genetic firewalls and crumbly DNA, we have a case where we have uracil rich plasmid, and via horizontal gene transfer, is transferred to another organism. And rather in the usual case whereby the organism confers this new function, the enzymes DUT and UNG present will actually recognize the uracil rich DNA and thus degrade it, preventing any spread of the DNA. In the other case of genetic missile and lethal mating, we have a genome which is rich in uracil and should it undergo mating with another organism, would take up synthetic DNA from the environment which could potentially contain these two enzymes, UNG and DUT, they will be expressed, hijacking the cell’s own polymerase functionality, and degrading the DNA. So, this method was coined in the de Lorenzo lab, whereby you take your gene of interest and you — denoted here by the orange arrows — and you choose regions upstream and downstream — denoted as TS1 and TS2 — to use as homologous regions later on, then for conventional methods such as PCR, we can create a whole entire fragment that has these two regions. Then, following on from restriction digest and subsequent ligation, we can integrate them into a plasmid which won’t replicate in our host organism, as it relies on a special polymerase and therefore, will actually integrate into the genome. Hijacking the cell’s own function of homologous recombination. And this works in such a way that when this plasmid enters, it will by homologous recombination, it will identify with the TS1 domain and will read through. And we’ll actually see the entire plasmid incorporated into the genome. And now, due to the presence of specific restriction enzymes’ sites in both the plasmid and in other parts of the genome, we can actually induce the response of an endonuclease to cleave these sites. Thus, giving us two possibilities by which we can recombine the DNA. We can have the first situation, which is where TS2 recombines with TS2, or we can have the other situation where TS1 recombines with TS1. Now this occurs with a 50/50 probability in the cell, and what we have is either a gene deletion variant through the homologous recombination of the TS2 domains, or we can have the wild type through the recombination of the TS1 domains. And this is achieved through a very standard array of methods applied in the lab such as PCR, restriction ligation, and digestion, and antibiotic selection. And so, it’s a rather novel method using a combination of fairly standard techniques, which is kind of what is at the heart of synthetic biology. And through creating these gene deletions variants and allowing the uracil rich incorporation into DNA, we can actually do some basic analyses and fluorescence microscopy, relying on propidium iodide staining of DNA to check for the presence of our DNA before and after the mating, and before and after the incorporation of these two enzymes. We can also do flow cytometry to look at a cell to cell basis, to see the actual ratio we’re getting of uracil incorporation. To see whether we get a uniform homogenous incorporation or whether there are actually changes that could affect the efficacy of this technique in creating genetic control. So, what we’ve done here is take native functions in an organism and actually repurpose them to create a means of genetic control to prevent the spread of genetically modified DNA adding to the environment. That’s really what’s at the heart of synthetic biology, is taking a negative function, redesigning it and repurposing it for our own means. Such that we could potentially improve our quality of life, save our environment, or help us to greater understand the intricacies of the world around us.