This genetic deletion is achieved through (i) heterologous recombination between the host chromosome and a suicide vector encoding a null allele and a resistance marker flanked by recombinase target sites, (ii) antibiotic-mediated selection for transformants, and (iii) subsequent excision of the resistance marker through transient expression of recombinase. risk can be minimized by increasing the stability of the genetic modification, both through chromosomal integration of the foreign DNA, and by ensuring that the chromosomal insertion site is distant from especially mobile genetic elements such as transposons. is Melagatran the possibility of releasing an organism with significant fitness advantage, leading to unrestrained propagation of the GMO and damaging environmental consequences. Fears of this outcome may be allayed through the use of auxotrophic strains unable to survive in the environment (i.e., biologically contained organisms) or through use of nonliving vehicles such as OMVs, BGs, and inactivated spores. is a risk unique to vaccine delivery: the possibility of an attenuated vaccine strain reverting to a virulent genotype, thus placing the vaccinee and/or close contacts in danger of contracting disease. Attenuation is usually accomplished by disrupting a gene involved with either virulence or central metabolic pathways. The former is associated with greater risk as virulence factors are generally located on mobilizable genetic elements that may be acquired from wild-type organisms. Metabolic genes, meanwhile, are generally located on the chromosome, so attenuations at these sites are more stable . The concernintroduction of drug resistance genes into the environmentis perhaps the most worrisome. In the laboratory setting, maintaining heterologous genes episomally in bacteria using antibiotic resistance selection markers (the simplest of recombinant techniques) is perfectly acceptable practice. However, it is imperative for genetically engineered bacteria released into the environmentsuch as vaccines and bioremediation devicesto be free of such markers, so as to prevent spread of drug resistance. Two genetic methods that satisfy this requirement have been established: the balanced lethal system (more widely used) ,  and recombineering both of which are effective in gram-negative and gram-positive  organisms. The uses an episomal plasmid to express the gene of interest, but importantly, the plasmid is maintained through complementation rather than antibiotic resistance , . Generation of a markerless carrier strain using the balanced lethal system proceeds as follows. A key metabolic gene such as one involved in amino acid synthesis is deleted, rendering the strain auxotrophic. This genetic deletion is achieved through (i) heterologous recombination between the host chromosome and a suicide vector encoding a null allele and a resistance marker flanked by recombinase target sites, (ii) antibiotic-mediated selection for transformants, and (iii) subsequent excision of the resistance marker through transient expression of recombinase. (serovar Typhimurium, and AIDA-I (adhesin involved in diffuse adherence) from to components of the gram-positive cell wall (Figure 4.1B,iv) , , , , . The key advantage of this methodology is that autolysin-passenger chimeras may be expressed in a model organism (e.g., and genera, display a unique behavior known as sporulation, in which bacteria transform from metabolically active (vegetative) cells into dormant structures (spores) in response to nutrient depleted conditions . During this process, bacterial DNA becomes cocooned within a multilayered wall of peptidoglycan and concentric protein shells, rendering the DNA impervious to toxic chemicals, radiation, and extremes of pH, temperature, TSPAN11 and dehydration . Spores are thus able to survive in inhospitable environments for extended periodsin one report, 25 million years , . Despite the spores extraordinary inertness, its surface proteins remain functional (in part due to their attachment to a stable matrix, see Section 4.3.4), ready to sense nutrient repletion, and germinate Melagatran into a fully active state . Numerous methods have been established for displaying heterologous proteins on these remarkable structures, all involving fusion to carrier proteins expressed at the spore surface . These carriers include CotB, CotC, and CotG in toxin ClyA, which becomes concentrated on these structures during vesiculation . OMVs have particular utility as vaccine delivery vehicles because they are stable during prolonged storage , and strongly immunogenic (by virtue of the many immunostimulatory components of the OM these vesicles retain), but do not present the same environmental safety hazards as intact cells because they are unable to Melagatran propagate (see Box 4.1) , , . BGs, in contrast, are complete gram-negative bacterial envelopes produced through heterologous expression of the bacteriophage-derived lysis gene (Figure 4.2 ). Upon expression, Protein E inserts into the IM via its N-terminus, while the C-terminal domain translocates across the IM (Figure 4.2A) and integrates into the OM (Figure 4.2B), inducing fusion of the two membranes. Protein E oligomerization leads to the.