The Antibiotic Epidemic Antibiotic Resistance

Antibiotic Resistance: Surviving An Uncertain Future

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Bt Transgenic Plants Are Big Business

Transgenic (genetically engineered) plants, scientists first identified the gene in Bt that codes for the toxic chemical. Once the gene is known, the techniques of genetic engineering can be used. The gene is removed from the Bt genome using specialized chemicals. It is then combined with another gene that codes for a chemical that degrades a particular antibiotic. This gene combination is then inserted into plant cells, where it is taken into the plant's DNA. As the plant grows, its new cells express the chemical as directed by the transplanted gene combination. When grown in the presence of the antibiotic, the plant cells that are resistant because they produce the Bt antibiotic-resistance protein can be identified. (Not all the plants take in the transplanted DNA effectively.) These cells are selected and grown into the Bt-expressing (producing) plants.

Scientific Background to Mer Operon

The Mer Operon is found in a very similar form in many bacteria because it exists in the form of a plasmid (a short section of circular DNA) on transposons (a mobile piece of DNA) and in cellular DNA, the genome of an organism. Plasmids, in particular, are frequently transferred between different species of bacteria as well as within the same species. This is one of the mechanisms by which antibiotic resistance spreads.

The beginnings of gene therapy

The plan was to splice a bacterial gene for resistance to a particular antibiotic into a virus, then culture the engineered virus with TILs. The virus would infect the TILs and transfer its genes onto their chromosomes. As the TILs later multiplied in culture, each would carry the telltale gene of antibiotic resistance. With this added marker, the TILs and their descendants could be tracked in tissue samples anywhere, anytime they would be the only cells that survived when soaked in the antibiotic. Finding engineered cells among a culture of hundreds of thousands of regular cells is like looking for a needle in a haystack. A common technique is to attach a genetic marker to the recombinant DNA. A bacterial gene for antibiotic resistance was used as a marker to track engineered tumor-infiltrating lymphocytes (TILs) In the first experiment in gene transfer to humans. gene for antibiotic resistance is clipped out Finding engineered cells among a culture of hundreds of thousands of...

Description of luxAB luxCDE and luc Vectors

We have constructed a number of vectors that contain the promoterless V. harveyi luxAB genes, which encode the luciferase enzyme, and an antibiotic-resistance marker, flanked by the sequences from one of two identified neutral sites of the cyanobacterial genome (Fig. 1). These neutral sites are regions of the S. elongatus chromosome that can be disrupted without any discernable phenotype (neutral site I, NS1, GenBank accession number U30252 neutral site II, NS2, GenBank accession number U44761). Vectors for NS2 target two adjacent regions of the chromosome (NS2.1 and NS2.2) based on the location of the antibiotic resistance cassette and cloning site for insertions within neutral site DNA on the plasmid. Because these vectors can replicate in E. coli but not in S. elongatus, a homologous recombination event (an apparent double crossover in which the trans-gene and selective marker are inserted into the neutral site and the vector sequence is lost) must occur between the neutral site...

Homologous Recombination at Neutral Sites of S elongatus Chromosomes

The basic principles underlying recombination at the NSs are the same as those described for gene replacement (see Subheading 3.1.). In this case, the sequence to be inserted is directed to one of these specific sites in the S. elongatus chromosome by cloning it within NS sequences in specialized plas-mids called neutral site vectors. (For details about the NS vectors, including a list of those vectors made by our lab and sample maps, refer to Chapter 8.) In brief, these vectors contain an antibiotic-resistance marker and a multiple cloning site where a gene of interest can be inserted, flanked by NS sequences from either NS1 or NS2. After introduction of plasmids into S. elongatus, the resulting recombinant clones are isolated by selection on media containing the proper antibiotic. Since they were developed, our laboratory has used S. elongatus NSs to introduce numerous kinds of engineered genes. Here, we describe some of these applications.

Mutagenesis by Homologous Recombination

By this method, any gene on the chromosome of S. elongatus can be replaced by a modified homologous allele (Fig. 1A-D). When inactivating a cyanobacterial gene, the recombinant allele of interest is constructed in an E. coli vector (which will not replicate in the cyanobacterium) by inserting an antibiotic-resistance cassette or a transposon into the coding region of the gene, making sure that the insertion is flanked on each side by at least 300 base pairs of homologous genomic DNA ( 2,9 see Note 3 and Fig. 1A). The recombinant mutant allele is introduced into S. elongatus by transformation or conjugation and subsequently crosses into the cyanobacterial genome by homologous recombination. Selection for a double crossover event and subsequent segregation of mutant chromosomes (because S. elongatus maintains multiple copies of its chromosome 10 ) is based on growth of the strain on media containing the antibiotic to which the interrupting cassette confers resistance. a Gene motivation...

Introduction of Foreign DNA Molecules into Bacteria

To ensure the survival and propagation of foreign DNAs, they must be inserted into a vector that can replicate inside bacterial cells and be passed on to subsequent generations of the bacteria. The vectors used for cloning are derived from naturally occurring bacterial plasmids or bacteriophages. Plasmids (page 74) are small circular DNA molecules found within bacteria. Each contains an origin of replication (page 88) and thus can replicate independently of the bacterial chromosome and produce many copies of itself. Plasmids often carry genes that confer antibiotic resistance on the host bacterium. The advantage of this to the scientist is that bacteria containing the plasmid can be selected for in a population of other bacteria simply by applying the antibiotic. Those bacteria with the antibiotic resistance gene will survive, whereas those without it will die. Figure 7.2 shows

Safety Concerns

Besides the safety concerns for caretakers, potential environmental spread of the vector should also not represent a hazard. Therefore, the FDA strongly discourages the presence of any antibiotic-resistance genes in the final vector. The recombinant antigen needs to be maintained stably either on a plasmid or integrated into the bacterial chromosome without antibiotic selection. As these concerns will be raised whenever a bacterial vector is moved into clinical applications, vector design should address them early in the preclinical phase.

Protecting consumers

Another health concern is that transgenic food carrying marker genes for antibiotic resistance might transfer the resistance to consumers eating the food. This raises the risk that resistant genes might be incorporated into germs, against which we would then have no defense. To reduce the use of antibiotic genes, researchers are developing other genetic markers, based on such things as color change, or ability to use certain sugars.

Herpes Simplex Virus

HSV amplicon vectors represent an alternative to replication-defective recombinant genomic vectors. The amplicon is essentially a eukaryotic expression plasmid that contains the following genetic elements (1) HSV-derived origin of DNA replication (ori) and packaging sequence ( a sequence) (2) transcriptional unit typically driven by the HSV-1 immediate early promoter or an alternative promoter followed by an SV-40 polyadenylation site and (3) a bacterial origin of replication and an antibiotic resistance gene for propagation in Escherichia coli (30,31). Even so, amplicon plasmids are dependent on helper virus for the replication machinery and structural proteins that are necessary for packaging the amplicon-vector DNA into virus particles. Recently, helper virus-free amplicon packaging methods were developed by using complementary overlapping cosmids or bacterial artificial chromosomes (BACs) (32-34). The use of such amplicons simplifies vector construction and minimizes the potential...