Genetic Safeguards to Prevent Horizontal Gene Transfer

00:00:11.16 Hi, my name is Jason Whitfield and as part of a
00:00:13.22 team here at EMBL for Synthetic Biology in Action
00:00:16.01 project, I'll be taking you through the genetic firewalls
00:00:19.20 to horizontal gene transfer.
00:00:20.26 In an effort to greater understand synthetic biology
00:00:22.29 and its emerging contributions to society.
00:00:26.00 So, synthetic biology is a field that is marrying
00:00:30.00 engineering, chemistry, and biology,
00:00:32.02 building on efforts of people who've come before us
00:00:35.01 in biology through the creation of sequencing
00:00:37.08 and the Genomics Project, to help us create
00:00:39.04 new to nature functions whereby we can help
00:00:42.01 microbes such as Pseudomonas putida
00:00:44.28 create things such as biofuels. To help us lessen
00:00:47.29 our reliance on fossil fuel methods that we currently use.
00:00:51.04 Also to create therapeutic drugs
00:00:53.06 to help us dispense with the messy and often time-consuming
00:00:56.04 and expensive methods that organic chemistry
00:00:58.08 has given us. Third, we can also create
00:01:00.20 biosensors, whereby we can actually selectively
00:01:03.14 look for a molecule of interest in a biological system
00:01:06.06 or even in the environment.
00:01:08.07 And lastly, or more importantly, we can
00:01:10.14 in fact do bioremediation, whereby we can
00:01:12.22 clean up some of the pollution that has accrued
00:01:15.00 over man's occupation.
00:01:16.26 To do this, we need to be able to contain
00:01:19.28 these genetically modified organisms that
00:01:21.20 we're creating. The Asilomar Conference of 1975
00:01:24.20 highlighted this, and since then a lot of genetic and physical
00:01:27.24 containment strategies have been put into place.
00:01:29.22 And these largely rely on the idea that
00:01:31.19 we keep bacteria in a lab or in a reactor.
00:01:34.13 However, bacteria can transfer their DNA.
00:01:37.13 This is a process known as horizontal gene transfer.
00:01:40.07 This is when DNA from one organism is transferred
00:01:43.05 to another, conferring that new function
00:01:45.16 to the recipient organism.
00:01:47.07 However, DNA can survive in the environment
00:01:50.00 for months in the right conditions. So even though
00:01:52.11 an organism is dead, it's DNA will still survive.
00:01:55.05 And this in fact can be taken up also.
00:01:57.17 This really highlights the idea that we need to create
00:02:00.07 genetic barriers to this uptake or conferrence of
00:02:03.25 genetically modified function.
00:02:05.11 This really highlights the need for us to create a
00:02:07.29 genetic control mechanism by which we can
00:02:10.02 prevent the spread of this DNA.
00:02:11.17 To do this, we need to go to our basic biology.
00:02:14.14 And this involves looking at DNA replication, which
00:02:16.29 is a high fidelity process involving the concerted and
00:02:20.02 complex interplay of a variety of enzymes and
00:02:22.14 proteins to create conventional DNA replication.
00:02:26.16 And this however is not always true.
00:02:29.12 And sometimes mistakes can occur whereby
00:02:31.19 uracil can be misrecognized and therefore misincorporated
00:02:34.23 into the DNA strand as thymine.
00:02:37.06 And this occurs through a variety of mechanisms.
00:02:39.25 Most often governed by the ratio pool of uracil
00:02:44.16 to thymine. However, there are enzymes
00:02:47.12 involved in the cell that can actually regulate this process.
00:02:50.19 DUTP nucleotide hydrolase, and this one, what it
00:02:56.12 does is actually converts UTP to the monophosphate
00:02:59.24 variant, liberating pyrophosphate and doing so,
00:03:02.23 shuttles it down the biosynthetic pathway
00:03:05.05 towards thymine formation. Thus, lowering the pool
00:03:08.11 and lowering its incorporation. Another
00:03:10.14 process the cell has in its proofreading is UTP
00:03:13.06 uracil DNA glycosylase. This enzyme
00:03:16.12 moves along the DNA strand and flips out nucleotides,
00:03:19.25 assessing their identities, and when it finds a uracil,
00:03:22.22 it cleaves the backbone bonds, thus initiating the
00:03:26.10 uracil repair response. So another mechanism
00:03:29.28 that can also occur is the deamination of cytosine
00:03:33.04 to the uracil, thus increasing the pool of uracil
00:03:36.08 present in the cell and level of misincorporation.
00:03:38.22 So, one of the kind of ethos of synthetic biology
00:03:44.16 is to hijack these native functions and repurpose them.
00:03:47.19 And so, the idea for this project is to hijack this misincorporation
00:03:52.17 process as a means of creating a genetic control
00:03:54.11 in the organism, Pseudomonas putida.
00:03:56.06 To kind of create a new genetic language,
00:03:58.20 whereby we have a genetic firewall that ensures
00:04:02.00 that if a plasmid is present in an organism, it will be rich
00:04:04.12 in uracil and its survival will be dependent on
00:04:07.02 it staying in that target host. And conversely,
00:04:09.23 creating an organism that has a brittle genome
00:04:12.13 which will accumulate these uracils and should
00:04:15.01 it come into contact through mating or other
00:04:17.17 methods with the copies of DUT and UNG,
00:04:20.16 it will in fact be degraded.
00:04:22.05 To look at the first example of these genetic
00:04:25.04 firewalls and crumbly DNA,
00:04:27.04 we have a case where we have uracil
00:04:29.04 rich plasmid, and via horizontal gene transfer,
00:04:31.20 is transferred to another organism. And rather
00:04:34.08 in the usual case whereby the organism
00:04:36.11 confers this new function, the enzymes DUT
00:04:39.00 and UNG present will actually recognize
00:04:41.07 the uracil rich DNA and thus degrade it,
00:04:44.11 preventing any spread of the DNA.
00:04:46.04 In the other case of genetic missile
00:04:48.16 and lethal mating, we have a genome
00:04:51.14 which is rich in uracil and should it undergo mating
00:04:54.12 with another organism, would take up synthetic DNA
00:04:57.22 from the environment which could potentially contain
00:04:59.18 these two enzymes, UNG and DUT, they will be expressed,
00:05:03.19 hijacking the cell's own polymerase functionality,
00:05:06.20 and degrading the DNA.
00:05:08.12 So, this method was coined in the de Lorenzo lab,
00:05:12.25 whereby you take your gene of interest
00:05:15.23 and you -- denoted here by the orange arrows --
00:05:18.26 and you choose regions upstream and downstream
00:05:21.07 -- denoted as TS1 and TS2 --
00:05:23.07 to use as homologous regions later on, then for
00:05:27.13 conventional methods such as PCR, we can
00:05:30.14 create a whole entire fragment that has these two
00:05:32.26 regions. Then, following on from restriction
00:05:36.28 digest and subsequent ligation, we can
00:05:39.14 integrate them into a plasmid which won't
00:05:41.20 replicate in our host organism, as it relies
00:05:43.24 on a special polymerase and therefore,
00:05:45.19 will actually integrate into the genome. Hijacking
00:05:47.28 the cell's own function of homologous
00:05:50.19 recombination. And this works in such a way
00:05:54.16 that when this plasmid enters, it will
00:05:57.17 by homologous recombination, it will identify
00:05:59.18 with the TS1 domain and will read through.
00:06:02.29 And we'll actually see the entire plasmid incorporated
00:06:05.02 into the genome. And now, due to the presence
00:06:08.19 of specific restriction enzymes' sites
00:06:11.08 in both the plasmid and in other parts of the genome,
00:06:14.20 we can actually induce the response of an
00:06:17.23 endonuclease to cleave these sites.
00:06:19.05 Thus, giving us two possibilities by which
00:06:21.26 we can recombine the DNA. We can have
00:06:25.11 the first situation, which is where TS2 recombines
00:06:28.20 with TS2, or we can have the other situation
00:06:30.19 where TS1 recombines with TS1.
00:06:32.20 Now this occurs with a 50/50 probability
00:06:35.05 in the cell, and what we have is either a gene
00:06:38.10 deletion variant through the homologous recombination
00:06:41.04 of the TS2 domains, or we can have the wild type
00:06:43.19 through the recombination of the TS1 domains.
00:06:45.18 And this is achieved through a very standard
00:06:47.22 array of methods applied in the lab
00:06:50.23 such as PCR, restriction ligation, and digestion,
00:06:54.01 and antibiotic selection. And so, it's a rather
00:06:56.13 novel method using a combination of fairly
00:06:59.29 standard techniques, which is kind of what
00:07:01.02 is at the heart of synthetic biology.
00:07:02.26 And through creating these gene deletions
00:07:05.06 variants and allowing the uracil rich incorporation into
00:07:07.26 DNA, we can actually do some basic analyses
00:07:10.12 and fluorescence microscopy, relying on propidium
00:07:13.17 iodide staining of DNA to check for the presence
00:07:16.02 of our DNA before and after the mating, and before
00:07:18.22 and after the incorporation of these two enzymes.
00:07:21.00 We can also do flow cytometry to look at a cell
00:07:24.14 to cell basis, to see the actual ratio we're getting
00:07:27.20 of uracil incorporation. To see whether we get
00:07:29.13 a uniform homogenous incorporation or whether
00:07:32.16 there are actually changes that could affect
00:07:34.04 the efficacy of this technique in creating
00:07:37.16 genetic control. So, what we've done here
00:07:40.22 is take native functions in an organism
00:07:43.08 and actually repurpose them to create a means of
00:07:46.20 genetic control to prevent the spread of
00:07:48.12 genetically modified DNA adding to the environment.
00:07:51.13 That's really what's at the heart of synthetic
00:07:53.05 biology, is taking a negative function, redesigning
00:07:55.28 it and repurposing it for our own means.
00:07:58.08 Such that we could potentially improve
00:08:00.11 our quality of life, save our environment, or help us
00:08:03.00 to greater understand the intricacies of the world around us.

This Talk

Audience:

Researcher

Recorded: June 2015

Talk Overview

Synthetic biology can be used to create biofuels, therapeutics, biosensors, and bioremediation tools. This often involves introducing new DNA into an existing organism. However, at the same time, it also is important to develop genetic safeguards to ensure that this new DNA is not unwillingly transferred to another organism. One safeguard is to create an organism lacking key DNA repair enzymes (uracil-DNA-glycosylase, UNG; dUTP pyrophosphatase, DUT) that are responsible for fixing misincorporated uracil in the genome. Organisms lacking DNA repair enzymes will accumulate uracil mutations in its genome, and, if it mates with another organism, the uracil mutated genome will be recognized as faulty and destroyed. The Synthetic Biology in Action participants describe these different safeguarding mechanisms and how they created a host organism with DNA repair enzymes deletions.

About the Speaker

Yu Heng Lau, Post-doctoral scholar at Harvard Medical School

Roberto Ferro, PhD student at the Technical University of Denmark

Dario Neves, PhD student at RWTH Aachen University

Jason Whitfield, PhD student at the Australian National University, Canberra

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