CH391L/S13/Biocontainment

Biocontainment

Biocontainment is a set of strategies to confine pathogenic and genetically modified organisms to the laboratory. Biocontainment is a concern in medical, agricultural, and synthetic biology operations. With many companies performing synthetic biology research and production biocontainment is of increased concern, as more and more exotic GMO’s are produced. Presently, there are no differences between biocontainment strategies for genetically modified organisms (GMOs) than there are for synthetic biology. Typically biocontainment is a mixture of physical barriers as well as an emerging field of genetic barriers placed on an organism to prevent it from entering the environment. Physical barriers as part of a biocontainment strategy are a part of general lab biosafety practices.

History of biocontainment for GMOs

The first use of a genetic barrier for a strategy of biocontainment was reported in 1987. The hok gene (encoding for a lethal toxin) was incorporated on a plasmid, this strategy prevents the organism from being viable when leaving the lab, and the plasmid cannot be incorporated in to other organisms, as it is encoding the toxin gene [1].

In 1993 the NIH produced a set of guidelines for research involving recombinant DNA molecules, in the report it is recommended that engineered microbe survival or engineered DNA transmission is less than 1 cell per 10^8 cells [2]. Currently, many genetic barrier strategies are able to reduce genetic transmission or provide induced lethality within these guidelines. A great number of instances of GMOs escaping from the lab have been observed [3]. However, there have been no bio-hazardous accidents linked to either GMOs or synthetic biology [4].

Containment strategies

Genetic safeguard strategies. Recombinant DNA (bright green) is introduced into the host chromosome (white wavy lines). Two pathways for engineered auxotrophy (A,B) kill synthetic organisms (blue) once they lose access to a supplement (+) in a controlled environment. The supplement either (A) suppresses a toxic gene product (−) or (B) provides nutrition to compensate for a genetic deletion (red X). The induced lethality system (C) produces a toxic gene product (−) in response to an inducer (i) such as IPTG, sucrose, arabinose, or heat. Gene-flow prevention (D) is accomplished by placing a toxic gene into the recombinant DNA (dark blue/bright green circle) in an immune host. Transfer of the recombinant plasmid kills unintended host cells [3].

Engineered Auxotrophy

Auxotrophy is the inability of an organism to synthesize a particular organic compound required for its growth. In synthetic biology this is used to create an organism that cannot grow when it leaves the lab or a production facility, because it would be unlikely to encounter the necessary metabolite in nature. This has been a strategy in research for over 70 years, made famous in Beadle and Tatum's Nobel prize-winning work on the one gene-one enzyme hypothesis.

Examples of engineered auxotrophy

• 1991 Conditional-Suicide Containment System for Bacteria Which Mineralize Aromatics – Here they have a Pseudomonas that degrades benzoates for bioremediation, and then dies when substrate is no longer present [5].

• 2003 Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10 – Here they have engineered a Lactococcus that produces il-10 for the treatment of Crohn’s disease, this strain requires thymidine for full viability as it has a knockout of thyA [6].

Induced Lethality

Induced lethality is a biocontainment strategy in which GMOs are contained by the induction of toxic genes upon leaving the lab setting. The toxin/antitoxin systems of bacteria have been used in many papers examining the use of induced lethality. This induction is typically controlled by the presence or absence of an induction molecule, but theoretically any inducible promoter can be adapted to this purpose. More recently, the idea of induced lethality that has been engineered to cause death after a certain number of cell cycles has been proposed [3]. A study was able to count events in a cell using a a riboregulated transcriptional cascade and a recombinase-based cascade and by coupling [7]. These counters could count cell cycle events and a toxin gene could be induced upon a certain number of cell cycles [3]. The 2012 synthetic biology class went into toxin/antitoxin systems in greater detail (http://openwetware.org/wiki/CH391L/S12/ToxinAntitoxins).

Examples of Induced Lethality

• 1991 - Development of efficient suicide mechanisms for biological containment of bacteria. – They use a lactose inducible system for the production of the RelF [8].

• 1994 - A conditional suicide system in Escherichia coli based on the intracellular degradation of DNA. – They used a heat inducible system paired with a nuclease gene [9].

Gene-Flow barriers

Gene-flow barriers are required to prevent the transmission of genetic material from the original host organisms to organisms outside of the lab. These systems typically have a toxin gene or nuclease that the host is immune to, but when the transgenic DNA enters another host organism it will result in death. In plants, recombinant DNA can be sequestered in the chloroplasts resulting in another physical barrier. DNA integrated in chromosomes should have a lower likelihood of transmission between species than those contained on plasmids.

Example of gene-flow barriers

• 2003 - A dual lethal system to enhance containment of recombinant micro-organisms. - In this paper they had the gene with the trait they wanted flanked by two toxin antitoxin systems [10].

Orthogonal genetics

Orthogonal genetic strategies for biocontainment (semantic containment) strategies include using Xeno Nucleic Acids (XNAs), which are unnatural nucleic acids that could operate in the same capacity as DNA, for encoding a desired protein or rearranging codons between canonical amino acids. This is an active area of research in synthetic biology but no real world biocontainment strategy using these methods has been deployed as of yet.

Success of various biocontainment strategies

Reported frequencies of engineered bacteria that escape various genetic safeguard systems. A 2-liter volume is represented here as a standard soft drink container (left). Lowest reported frequencies (shown on the y-axis, log scale) were multiplied by the estimated number of cells in 2-liters at 1 × 108 cells/mL, where OD600 = 0.1 [BioNumbers record ID 10985 (Milo et al., 2009)]. The dashed line indicates the maximum survival limit (1000 cells per 2 liters) recommended by the National Institutes of Health (Wilson, 1993) [3].

Concerns about genetic biocontainment strategies

Genetic safeguards for preventing organisms from entering the environment are subject to spontaneous mutations and can cause failure to many biocontainment strategies. Many strategies are developing that contain multiple genetic barriers.

An illustration of the accumulation of damaged genetic safeguards in a population of synthetic organisms. When cells with intact safeguards (blue) escape physical containment (e.g., an accidental spill), an inducer (i) can be added to remove them from the environment. As the population grows, leaky expression of the lethal protein (−) reduces the viability of cells that carry functional safeguards. Mutation (X) of the lethal gene provides a growth advantage, thus cells that carry damaged safeguards (red) overwhelm the population. Cells with mutated safeguards do not respond to the cell death inducer (i). Consequently, it is difficult to remove the cells from the environment after an accidental release. [3].

Outreach about Synthetic Biology and Biocontainment

The Woodrow Wilson Synthetic Biology Project was developed to establish a public and policy discourse about synthetic biology. The project makes an effort to discuss biosafety involved with synthetic biology. They recently published a paper on biosafety and synthetic biology and what they believe needs to be further researched [11].

The four steps from the paper:

• 1) How can synthetic organisms' physiology disrupt nature?
• 2) How can synthetic organisms disrupt habitats?
• 3) Can synthetic organisms evolve quickly and persist in habitats?
• 4) How much risk is associated with gene transfer?

IGEM Connection

The 2012 UCSF IGEM team used a toxin/antitoxin system to tune strain ratios in E. coli. The project overview is described here: http://2012.igem.org/Team:UCSF/Toxin_System and the project results are described here: http://2012.igem.org/Team:UCSF/Toxin_Data

References

<biblio>

1. Haynes2013 pmid=23355834
2. Lorenzo2012 pmid=22710182
3. Dana2012 pmid=22382962
4. Contreras1991 pmid=16348490
5. Steidler2003 pmid=12808464
6. Knudsen1991 pmid=2036024
7. Ahrenholtz1994 pmid=7986048
8. Torres2003 pmid=14663091
9. Molin1987 (Molin et al., 1987) http://www.nature.com/nbt/journal/v5/n12/abs/nbt1287-1315.html
10. Wilson1993 pmid=11652293
11. Friedland2009 pmid=19478183