Biocontainment Systems Research Articles

Engineering a bacterial "Deadman" walking

Synthetic Biology One of the most successful areas of synthetic biology is modifying microorganisms for applications in biotechnology. However, these engineered microbes could pose a risk if released into open environments. To overcome this, Chan et al. engineered two modular gene circuits into the bacteria Escherichia coli that act as kill switches. The “Deadman” circuit requires a specific small molecule to prevent a toxin from being expressed and killing the cell. In the “Passcode” circuit, small molecules must induce two transcription factors to inhibit toxin expression. An advantage over other biocontainment systems is that these circuits can be reprogrammed to respond to different inputs or to induce different killing mechanisms. Nat. Chem. Biol. 10.1038/NCHEMBIO.1979 (2015).

'Deadman' and 'Passcode' microbial kill switches for bacterial containment.

Biocontainment systems that couple environmental sensing with circuit-based control of cell viability could be used to prevent escape of genetically modified microbes into the environment. Here we present two engineered safe-guard systems: the Deadman and Passcode kill switches. The Deadman kill switch uses unbalanced reciprocal transcriptional repression to couple a specific input signal with cell survival. The Passcode kill switch uses a similar two-layered transcription design and incorporates hybrid LacI/GalR family transcription factors to provide diverse and complex environmental inputs to control circuit function. These synthetic gene circuits efficiently kill Escherichia coli and can be readily reprogrammed to change their environmental inputs, regulatory architecture and killing mechanism.

Transgene autoexcision in switchgrass pollen mediated by the Bxb1 recombinase.

BackgroundSwitchgrass (Panicum virgatum L.) has a great potential as a platform for the production of biobased plastics, chemicals and energy mainly because of its high biomass yield on marginal land and low agricultural inputs. During the last decade, there has been increased interest in the genetic improvement of this crop through transgenic approaches. Since switchgrass, like most perennial grasses, is exclusively cross pollinating and poorly domesticated, preventing the dispersal of transgenic pollen into the environment is a critical requisite for the commercial deployment of this important biomass crop. In this study, the feasibility of controlling pollen-mediated gene flow in transgenic switchgrass using the large serine site-specific recombinase Bxb1 has been investigated.ResultsA novel approach utilizing co-transformation of two separate vectors was used to test the functionality of the Bxb1/att recombination system in switchgrass. In addition, two promoters with high pollen-specific activity were identified and thoroughly characterized prior to their introduction into a test vector explicitly designed for both autoexcision and quantitative analyses of recombination events. Our strategy for developmentally programmed precise excision of the recombinase and marker genes in switchgrass pollen resulted in the generation of transgene-excised progeny. The autoexcision efficiencies were in the range of 22-42% depending on the transformation event and assay used.ConclusionThe results presented here mark an important milestone towards the establishment of a reliable biocontainment system for switchgrass which will facilitate the development of this crop as a biorefinery feedstock through advanced biotechnological approaches.

The Endurance Testing Process: A Methodology to Verify the Functionality of Biocontainment, Security, and Life Safety Systems in BSL-3 and BSL-4 Laboratories

Biocontainment facilities planning to possess or work with biological select agents and toxins (BSAT) require registration with the National Select Agent Registry. The registration process includes submission of documentation for review to either the Centers for Disease Control and Prevention (CDC) Select Agent Program or the U.S. Department of Agriculture (USDA) Animal and Plant Health Inspection Services, followed by a site inspection. The process serves to assess whether an entity meets requirements to safely and securely conduct work with BSAT. Part of the assessment focuses on the facility itself with regards to design, construction and engineering. Testing and verifying that facility systems integral to biocontainment and overall safety and security are functional and reliable is an essential prerequisite. The National Biodefense Analysis and Countermeasures Center (NBACC) developed a process of endurance testing to verify the facility could be operated in a safe and secure manner. Information prese...

Effective Animal Health Disease Surveillance Using a Network-Enabled Approach

There are many benefits that derive from real-time knowledge of the health status of the national livestock population. Effective animal disease surveillance is a requirement for countries that trade in live animals and their products in order to comply with the World Organization for Animal Health (OIE) guidelines. Rapid identification of introduced and emerging disease allows rapid response and mitigation of the economic consequences. Connections between animal and human disease caused by a common pathogen can be recognized and control measures implemented, thereby protecting public health and maintaining public confidence in the food supply. Production-limiting diseases can be monitored, and control programmes be evaluated with benefits accruing from decreased economic losses associated with disease as well as reducing the welfare concerns associated with diseased animals. Establishing a surveillance programme across a wide area with diverse ecosystems and political administrations as Canada is a complex challenge. When funding became available from a government programme to enable early detection of a bio-terrorist attack on livestock, the Canadian Animal Health Surveillance Network (CAHSN) became officially established. An existing web-based information platform that supports intelligence exchange, surveillance and response for public health issues in Canada was adapted to link the network animal health laboratories. A minimum data set was developed that facilitated sharing of results between participating laboratories and jurisdictions as the first step in creating the capacity for national disease trend analysis. In each of the network laboratories, similar quality assurance and bio-containment systems have been funded and supported, and diagnostic staff have been trained and certified on a suite of diagnostic tests for foreign animal diseases. This ensures that national standards are maintained throughout all of the diagnostic laboratories. This paper describes the genesis of CAHSN, its current capability and governance, and potential for future development.

Design and testing of a biocontainment system for chemical carcinogens.

A biocontainment facility was built for studies in which the chemical carcinogen N-methyl-N-nitrosourea (MNU) was instilled intrarectally in guinea pigs. The system operated by constant flow of uncontaminated air into carcinogen-contaminated animal isolation chambers and filtration through a high-efficiency particulate air (HEPA) filter prior to release into the environment. The facility was tested for efficiency of carcinogen containment by substituting for the MNU a similar concentration of a fluorescent tracer, sodium fluorescein, under standard operating procedures for carcinogen administration to guinea pigs. Wipe samples from the floor, isolation chambers, animal handlers and clothing, and intake and exhaust air samples were analyzed for fluorescein before and after intrarectal instillation of the tracer. The recovery of very low concentrations of total and respirable suspended fluorescein from sampling points within the facility and the absence of detectable fluorescein in the air downstream from the HEPA filter indicated that the facility provided adequate protection against contamination of personnel or the environment.