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| - | <h1>Introduction: high gene expression in industrial biotechnology</h1> | + | <tr> |
| - | <p>The main goal of industrial biotechnology is to increase the yield of biochemical products using microorganisms as production hosts. This includes engineering large synthetic pathways and improving their expression. Overexpression of genes has mainly been achieved by using high or medium copy plasmids. However, studies have demonstrated that plasmid-bearing cells lose their productivity fairly quickly as a result of genetic instability.</p> | + | <td> |
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| - | {{:Team:UGent/Templates/ToggleBoxStart}} Read more about plasmids {{:Team:UGent/Templates/ToggleBoxStart1}}{{:Team:UGent/Templates/ToggleBoxStart2}}
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| - | <p>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.</p>
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| - | <p>Therefore a new method was developed for the overexpression of a gene of interest in the bacterial chromosome: Chemically Inducible Chromosomal evolution (CIChE). In this technique the chromosome is evolved to contain a higher number of gene copies by adding a chemical inducer. The original model for CIChE, however, results in bacterial strains containing a large number of antibiotic resistance genes.</p>
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| - | {{:Team:UGent/Templates/ToggleBoxStart}} Read more about CIChE{{:Team:UGent/Templates/ToggleBoxStart1}}{{:Team:UGent/Templates/ToggleBoxStart2}}
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| - | <p>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 characteristics. 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.
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| - | CIChE works as follows: First a construct, containing the gene(s) of interest and the antibiotic marker chloramphenicol acetyl transferase (<i>cat<i>) 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 (<b>Figure<b>).
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| - | [[File:UGent_2013_CIChE.jpg|thumb|400px|center|Tyo et al., 2009]]
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| - | This process is called chromosomal evolution. When the desired gene copy number is reached, <i>recA<i> is deleted, thereby fixing the copy number.</p>
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| - | <p>To make this valuable technique more widely applicable in the industry, we developed a model for chromosomal evolution based on a toxin-antitoxin system instead of antibiotic resistance.</p>
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| - | {{:Team:UGent/Templates/ToggleBoxStart}} Read more about antibiotic resistance{{:Team:UGent/Templates/ToggleBoxStart1}}{{:Team:UGent/Templates/ToggleBoxStart2}}
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| - | <p>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.
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| - | 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.
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| - | 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.</p>
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| - | <h1>Our model</h1>
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| - | <h1>Toxin-Antitoxin systems</h1>
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| - | <p>In our opinion it is possible to eliminate the need for antibiotics by using a toxin-antitoxin system. These systems are widely distributed among bacteria and archaea. The toxins produced by these systems can slow down or stop cell growth by interfering with certain molecules that are essential in cellular processes like DNA replication, cell wall synthesis, ATP synthesis etc.. Under normal conditions, the toxin is inhibited by the antitoxin, which is encoded in the same operon as the toxin. Research revealed different types of TA systems.</p>
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| - | {{:Team:UGent/Templates/ToggleBoxStart}} Read more about the different types of TA systems {{:Team:UGent/Templates/ToggleBoxStart1}}{{:Team:UGent/Templates/ToggleBoxStart2}}
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| - | ''' Type I'''
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| - | 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.
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| - | '''Type II'''
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| - | 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.
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| - | '''Type III'''
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| - | 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.
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| - | ==== CcdA/CcdB ====
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| - | This type II TA system is the first identified and best studied of all TA systems, which is why we have chosen to use it in our model. The target of the CcdB protein is the A subunit of DNA gyrase. This gyrase is an essential type II topoisomerase, occurring in all bacteria but not in eukaryotes. Bacterial gyrases have the property to introduce negative supercoils into DNA, which makes them unique among topoisomerases.
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| - | In the absence of its antidote CcdA, CcdB causes reduced DNA synthesis, activation of the SOS pathway, cell filamentation and eventually cell death. When CcdB interacts with a gyrase:DNA complex, it stabilises the cleavable complex. In this mode of action, CcdB acts as a poison, promoting DNA breakage mediated by gyrase. The antidote CcdA can inhibit CcdB by the formation of a tight non-covalent complex45. This prevents CcdB from making a covalent complex with gyrase. But even after CcdB has formed a complex with gyrase, CcdA can still reverse the blocking CcdB caused
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| - | === Construct ===
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| - | [[File:UGent_2013_Model.jpg|thumb|700px|center|Our model for CIChE]]
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