In industrial biotechnology, a common technique to express new synthetic products and pathways is the use of plasmids as vectors. Plasmids are circular pieces of DNA. They 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: plasmid maintenance imposes a metabolic burden on cells and plasmids suffer from genetic instability.

Metabolic burden

When plasmids are present in cells and replicate, they create a metabolic burden. This is defined by Bentley et al. (1990) as “the amount of resources that are taken from the host cell metabolism for foreign DNA maintenance and replication”. As a result, metabolic load causes many alterations to the physiology and metabolism of the cell and reduces the cellular fitness. The most common change is a delayed growth. This can be caused by the fact that new pathways for energy generation are activated due to the competition between cell propagation and plasmid replication. This growth retardation leads to a lower yield of the desired product.

Genetic instability

Plasmids are genetically instable due to three processes: segregational instability, structural instability and allele segregation.

• Segregational instability is caused by unequal distribution of plasmids to daughter cells, which leads to cells devoid of any plasmids. This problem has been solved by using selectable markers (e.g. antibiotic resistance) or post-segregational killing to remove plasmid-lacking cells.
• Structural instability leads to an incorrect expression of proteins. This problem finds its cause in plasmids with a changed DNA sequence. However, these mutations occur at a low frequency.

Allele segregation up to now remains mainly unaddressed. When a mutation occurs in the gene of interest, but not in the selectable marker, cells can emerge that are resistant to the selection, but not productive. As a result they cannot be removed by the use of selectable markers or post-segregational killing (Figure). After the mutation occurs, the plasmids are replicated and divided over daughters cells. The mutated plasmids can be divided in two different ways: either each daughter cell receives one mutated plasmid, or only one daughter cell receives both. In the latter case, the cell receiving both mutated plasmids produces less of the desired product and grows faster than the other cell. This growth advantage is due to the fact that the new synthetic pathway that has been inserted places a heavy metabolic burden on cells and reduces the cellular fitness. Therefore, cells with mutated plasmids accumulate and lead to a great productivity loss.

Allele segregation results in a rapid loss of productivity in plasmids

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.

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

This process is called chromosomal evolution. When the desired gene copy number is reached recA is deleted, thereby fixing the copy number.

The use of CIChE-strains poses several advantages. They require no selection markers and can be cultured without the addition of antibiotics to the medium. This is in contrast to strains containing plasmids, which still need antibiotic selection. Also, in CIChE-strains yields can be tuned by varying the antibiotic concentration during chromosomal evolution.

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 it is very unlikely to occur the possibility of horizontal gene transfer cannot be entirely ruled out.

The fact that the strain eventually contains multiple antibiotic resistance genes withholds this CIChE technique from being applicable in for example food industry, given several regulations as well as consumer’s mind-set. 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.

Gene of interest

For our experiment, it would be useful to select a gene of interest that can easily be identified. Using a fluorescent reporter gene such as gfp is the obvious choice. When GFP is brought to expression in E. coli this results in the production of green light when illuminated with an ultraviolet source. A great advantage of GFP is the fact that no substrate is needed for its expression. Hence, GFP is ideal to measure gene expression, as it is not limited by substrate availability. The intensity of the fluorescence is proportional to the GFP presence in the cell, providing a first indication of the number of gfp genes present in the cell. Consequently, fluorescence intensity could give us a first insight in the number of construct copies attained through chromosomal evolution.

Inducible promoter for ccdB

To increase the gene copy number in the chromosome, we have to be able to regulate the amount of CcdB in the cell. To achieve this, an inducible promoter is used. An inducible promoter is a combination of an operator and a promoter. The operator may be switched on or off which controls the transcription of the gene. Sometimes it is possible to regulate transcription according to the amount of stimulus the promoter receives.

The T7 promoter is not recognised by the E. coli RNA polymerase. This allows for good regulation of the transcription of genes, because there is no transcription as long as there is no T7 RNAP present. By using an inducible promoter to regulate the production of T7 RNAP, it is possible to change the amount of transcription of the desired protein. This is usually done by an IPTG inducible promoter.

Other options we considered were the arabinose promoter and the lactose promoter. We chose the T7 promoter because it shows lower leaky expression than these two. Leaky expression is transcription that occurs when no stimulus is given to the promoter. For the transcription of a toxic protein as in our case, leaky expression has to be avoided because even a small amount of transcription can be a burden to the cell and restrict cell growth.

The T7 promoter can be induced by IPTG. By using different concentrations we try to find a correlation between the applied IPTG concentration and the amount of gene copy numbers in the genome.