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Promoters for the construction of genetic circuits

Gene switches, or promoters, are crucially important for the regulation of all cellular activities, and thus play a pivotal role for researchers constructing externally controllable genetic circuits using synthetic biology methods. The most common artificial regulatory system uses the Tet technology and tetracycline-sensitive promoters invented by Bujard and his team.

Controlled gene expression, i.e. the arbitrary switching on and off of specific genes through experimenters, is one of the most important methods in basic research for the investigation of gene function. It is also an essential tool in synthetic biology in order to specifically modify regulatory circuits and control them exogenously. Gene switches, with which the activity of specific genes can be specifically regulated by the addition or omission of molecules, therefore play a key role in the construction of genetic circuits. For example, they can be used in bioproduction processes to adapt the metabolic pathways of cells to the requirements of certain syntheses, or correct defective processes in cancer or genetic metabolic diseases using gene therapy approaches.

The Tet system

Prof. Dr. Dr. h.c. Hermann Bujard © University of Heidelberg

Nowadays, the most frequently used technology to control the expression of genes in higher organisms is the Tet technology developed by Gossen and Bujard and presented for the first time in 1992. The Tet technology is based on a genetic circuit that regulates the bacterial resistance to the antibiotic tetracycline (which inhibits bacterial protein biosynthesis by binding to the A site of ribosomes). The transcription of the tetracycline resistance operon is inhibited by the Tet repressor in the absence of tetracycline; transcription is induced upon the binding of tetracycline to the repressor. Bujard and his team were able to adapt this regulatory system to eukaryotic cells, enabling them to specifically switch genes in mammalian cells on and off. For example, in a human tumour cell line equipped with a Tet promoter, the  researchers were able to gradually increase the activity of a specific gene (titratable genes) to 250,000-fold (determined from the concentration of the gene product) by modifying the amount of tetracycline in the culture medium. The system is characterised by an extraordinary sensitivity.  The smallest traces of tetracycline in foetal calf serum (antibiotics are often used as animal growth promoters) used for cell culture can falsify the experiments. Three nanogrammes doxycycline per millilitre of culture, which is the most common tetracycline derivative used nowadays, are sufficient to completely block the promoter.

In order for a target gene to be expressed in eukaryotic cells, it needs to be kept under strict promoter control in order for it to be transcribed. In the case of the Tet system, the Tet repressor is fused with a viral activation domain and transferred into a transcription activator known as “tetracycline-dependent transactivator” (tTA), which is encoded by one of the two vectors used by the Tet system. The promoter of the other vector contains a multiple cloning site (polylinker) for the gene to be cloned, whose transcription is regulated by tetracycline. In the “Tet-off” variant of the system, gene expression is activated in the absence of tetracycline or doxycycline, whereas “Tet-on” contains a reverse tetracycline-dependent transactivator which activates gene expression only in the presence of the antibiotic.

More than 6,000 scientific papers have been published on the use of the Tet system. The technology is protected by a broad patent portfolio owned by the company TET Systems Holding GmbH & Co. KG in Heidelberg that was set up by Bujard and his colleagues. TET Systems Holding commercialises Tet technology licences through its subsidiary IP Merchandisers. More than 150 organisations, including large academic institutions, scientific foundations and pharmaceutical and biotech companies have acquired licences for their own research and development activities. The 150 organisations also include 17 of the 20 largest global pharmaceutical companies. The licence is free of charge for academic researchers when the technology is purchased from the company Clontech (now part of Takara) which has been in charge of the commercialisation of the Tet system ever since its discovery.

Modular regulatory systems

The majority of functions in cells and the majority of disease-inducing events are influenced by several or sometimes even quite a large number of genes. In order to gain insights into these processes and possibly also change them, several genes need to be regulated simultaneously and independently from each other. The huge advantage of the Tet system is that it can be adapted to numerous applications and that it has been further developed, modified and optimised for different functions. Several operator sequences are available that are specifically and selectively recognised by certain Tet repressor alleles. It is also worth noting that tissue-specific promoters are available, which are often of greater interest for medically oriented applications than ubiquitously active promoters.

Besides the Tet system, numerous other genetic circuits have been developed. One of these is the frequently used Cre/loxP system which can be used to specifically remove DNA sequences from living cells. In contrast to the Tet system, which is reversible and enables the repeated switching on and off of genes, Cre/loxP is irreversible. It can be envisaged that one day several regulatory systems will be combined with each other in a modular system (“Biobricks”) for use as synthetic biology applications. This can lead to complex cybernetic systems with positive and negative back-coupling whose interactions can only be predicted with difficulty, and hence need to be analysed in laboratory experiments. It is necessary for classical biotechnology, systems biology and synthetic biology to work closely together in the construction of complex genetic circuits.

International synthetic biology competition

In the laboratories of BioQuant, students from Heidelberg work with effortless ease on the construction of such genetic circuits for mammalian cells. “The work with mammalian cells is of particular importance for us here in Heidelberg, where our aim is to be able to extend synthetic biology methods to cancer research,” explained Professor Roland Eils from the German Cancer Research Centre and Heidelberg University, who coordinates the “Spybricks” project.

The Heidelberg team who won the iGEM competition 2009. © DKFZ

The "Spybricks" project team from Heidelberg consisted of 13 students who used a novel chemical synthesis method to generate different promoter sequences at random. The students subsequently evaluated the different promoters for their ability to activate genes and for their function in the cell. In addition, promoters that were able to attach to certain regulatory proteins were designed with computers and their function evaluated in laboratory experiments. The goal of the project was to create new gene switches, for example for cancer cells that only react to clearly defined signals. The sequences of all newly synthesised gene switches were stored in a library that serves as a repository which is open to all scientists working in the area of synthetic biology.

The team from Heidelberg led by Professor Eils participated in the highly prestigious International Competition of Genetically Engineered Machines (iGEM) organised by the Massachusetts Institute of Technology in Boston in November 2009. The Heidelberg team achieved an extraordinary second place in the overall evaluation; over 110 teams and more than 1,100 students of the world's best universities participated in the competition. The Heidelberg students also received a prize for the best new technical standard and best Internet representation. In 2008, the team participated in the competition for the very first time and won three special awards.

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/promoters-for-the-construction-of-genetic-circuits