Team:UGent/Project

In industrial biotechnology, a common technique to express new synthetic products and pathways is the use of plasmids as vectors. Plasmids are easy to insert into cells and replicate independently from the genome, allowing strong gene expression. Overexpression is easily achieved by using plasmids with a medium or high copy number, different promoter systems, ribosome binding sites (RBS), etc. Thanks to plasmids, the industrial biotechnology has grown substantially over the past years. However, the use of plasmids entails some important disadvantages.

In 2009, Tyo et al. developed a technique for the stable, high copy expression of a gene of interest in E. coli without the use of high copy number plasmids, thus avoiding their previously stated negative characteristics2. They called this plasmid-free, high gene copy expression system ‘chemically inducible chromosomal evolution’. In this method, the gene of interest is integrated in the microbial genome and then amplified to achieve multiple copies and reach the desired expression level. Genomic integration guarantees ordered inheritance, resolving the problem of allele segregation. CIChE works as follows: First a construct, containing the gene(s) of interest and the antibiotic marker chloramphenicol acetyl transferase (cat) flanked by homologous regions, is delivered to and subsequently integrated into the E. coli genome. The construct can be amplified in the genome through tandem gene duplication by recA homologous recombination. Then the strain is cultured in increasing concentrations of chloramphenicol, providing a growth advantage for cells with increased repeats of the construct and thereby selecting for bacteria with a higher gene copy number (Figure).

Tyo et al., 2009

Antibiotic resistance is the resistance of a bacterium to an antibacterial medicine a.k.a. antibiotic to which it was originally sensitive. When a pathogenic bacterium has acquired resistance to a certain antibiotic, infections of this bacterium can no longer be treated with the antibiotic in question since the bacterium has become insensitive to the antibiotic. Overuse and misuse of antibiotics has accelerated the emergence and spread of resistant bacteria. Today antibiotic resistance has become a fairly well-known term as a result of the recurring appearance of multi-drug resistant bacteria such as MRSA and VRE in media, awareness campaigns etc. These bacteria have become resistant to the commonly used antibiotics. As a result infections are very difficult to treat. The emergence of these hard-to-kill pathogenic bacteria poses a serious risk to public health.

In biotechnology antibiotic resistance genes (the genes that make a bacterium resistant to a certain antibiotic) are often used as selectable markers. The best known application is probably the selection of plasmids. In the original CIChE technique a chloramphenicol resistance gene is used as selectable marker. During chromosomal evolution the antibiotic resistance gene is duplicated since it is the driving pressure for the tandem duplication of the CIChE-construct. As a result the final bacterial strain carries multiple antibiotic resistance genes. The creation of such a strain raises several safety concerns as bacteria can pass resistance genes on to other, possibly pathogenic, bacteria in the environment through a process called horizontal gene transfer. Although this is very unlikely to occur the possibility of horizontal gene transfer cannot be entirely ruled out. We will try to replace the use of antibiotic resistance genes with a toxin-antitoxin system. By eliminating the need for antibiotic resistance genes, CIChE could be applied in the industry without having to worry about the possibility of horizontal gene transfer and the spread of antibiotic resistance.

Type I

The first category of TA systems is characterised by an antisense mechanism that regulates the expression of the toxin gene. This antisense RNA is transcribed from the same toxin region, but in reversed orientation, and encodes the antitoxin. This antitoxin anneals to the toxin mRNA and forms double-stranded RNA across the ribosome binding site. This induces the degradation of the toxin mRNA or the blocking of the ribosome binding site. As a result, the amount of toxin in the cell is reduced.

Type II

Antitoxins in type II TA systems are proteins and inhibit the corresponding toxin by forming a stable TA protein complex. These systems are encoded by an operon that contains the two genes encoding the toxin and antitoxin. The antitoxin has to be continuously produced, as it is less stable than the toxin. Because of this feature, the cell has a feedback mechanism to prevent growth arrest. The TA complex functions as a repressor that binds the promoter and thereby inhibits transcription. Thus, when the antitoxin is degraded and the concentration of the TA complex decreases, the TA operon is derepressed and produces more toxin and antitoxin. Finally, the concentration of the antitoxin is reestablished. The rank of the two genes differs, which can contribute to different toxin-antitoxin ratios.

Type III

In the last category of TA systems, an RNA antitoxin directly inhibits the toxin. These RNA antitoxins are pseudoknots, which contain internal stemloops. The toxin and its antidote are once more encoded by the same operon. A transcriptional terminator between the two genes, regulates the ratio of toxin and antitoxin in the cell.