Aim

Within NUTRInity-Provide, we aim to establish a shuttle system with countless applications. In our case, we want to produce micronutrients directly where the absorption in the human body takes place – in the small intestine. However, we do not want to release genetically modified microorganisms into our guts if it is not really necessary. Therefore, we want to find a system that can produce valuable compounds but is no longer able to divide. For this purpose, we want to establish a minicell production E.coli chassis and show that we can produce micronutrients like β-carotene. Furthermore, we built a modular system to transform any bacterial chassis into a minicell producer.

Project Design

In order to get a functional production shuttle in the human gut, we used the minicell-producing E.coli strain TB43 whose Min-gene system was deleted. We decided to utilize this kind of system to reduce risks of biosafety as far as possible for the minicells are not able to propagate. As comparison, we also tested a strain from the Keio collection with a deletion of MinC. We examined both strains via flow cytometry to evaluate minicell production. Originally, flow cytometry was developed for the examination of fluorescence in cells, which is why we had to optimize the measurement in order to differentiate between minicells and their parent cells. We decided to use both, size and complexity as distinctive features.

For the determination of the isolated minicells’ characteristics, we established a novel purification protocol using different filtration systems to get rid of chromosomal DNA containing cells. This was an important point in maintaining biosafety in terms of possible applications. Furthermore, we had to make sure that the minicells would not start dividing all of a sudden. For this reason, we performed a time laps experiment using fluorescence microscopy over several hours.

To see whether micronutrients can be produced by our chosen system, we transformed a plasmid encoding the four necessary genes (dxs, crtE, crtI and crtB) for the biosynthesis of β-carotene into the TB43 strain. β-carotene is a vital compound, which is necessary for the production of vitamin A in the human body. A lack can lead to a weak immune system or blindness[6] which is still common in many developing countries.

After successful production of this nutrient, we thought of a way to make this non-living chassis suitable for as many applications as possible. For this purpose, we needed to knock down one part of the Min-system. So, we designed a modular sRNA-encoding construct to target the MinC-mRNA to generate the needed knock down strain.

Results

Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua.

Outlook

As the chemical synthesis of many compounds is still a problem due to inefficient reactions, the need of chemicals produced by microorganisms has been aimed for over the last decade. Especially the production of pharmaceuticals is an important aspect. Microorganisms might also produce dietary supplements in the near future.

We want to deliver the minicells in form of a pill, which is resistant to stomach acid and dissolves as soon as it gets into the duodenum. There, the minicells will produce the micronutrients and excrete them into the intestines. We think that this is a better method for a successful supply of micronutrients in comparison to an increase of the concentration in food as the absorption of for example dietary β-carotene out of food is not very efficient. This is caused by strong binding to many different biomolecules due to inflexibility of its conjugated structure.

To ensure biosafety for both, the consumer and the environment, purification of the minicells has to be conducted. This prevents settlement of engineered bacteria in the human gut and their propagation after excretion. We mainly use different filters for the purification to get rid of ordinary cells on the one hand and to increase the concentration of the minicells on the other.

We want to make minicells suitable for everyone as they have the potential to act as a chassis with a lower risk compared to ordinary bacteria. Thus, they fit for multiple applications. Our idea is a sRNA encoding plasmid which silences MinC. With this plasmid, every strain could be brought to the production of minicells by a simple transformation.

To achieve all the described future goals, further experiments have to be conducted. Most of these experiments were planed but haven’t been achieved so far. The first step is to use FACS with a cell sorter to get a 100% separation of minicells from cells containing chromosomes. This needs to be ensured so that our further analytics are not contaminated by non-minicells. Our facility will get a cell sorter soon, which will allow us to proceed with this improved purification. The next step is to analyze the metabolic activity of the minicells using metabolic approaches. For the first proof we are going to test, if we can observe glycolysis in the minicells. Further more we are going to test if we can induce whole metabolic pathways e.g. the one for β-carotene in the purified minicells. If these tests are successful, we can use the minicells even for the production for growth inhibiting products as no cell proliferation is needed within the minicells.

Background

Bacterial minicells are produced by asymmetric cell division, which is caused by a knock out of the minCDE-system. Naturally, this system regulates symmetric cell division.^[1] The Min-proteins bind to the cell poles and oscillate between them. MinC functions as inhibitor for FtsZ, which forms the Z-ring and ultimately initiates division of the cell. This symmetric division ensures that each cell gets a copy of the chromosome and all other cell components and thus, is able to live on its own. If this system is disturbed, cell division occurs randomly leading to the formation of minicells.

As only a MinC knock down is needed to get minicells, we looked for a simple and modular solution. The knock down of gene expression through a small regulatory RNA (sRNA) system was already shown in iGEM by the Team Paris Bettencourt 2013 which was a great success for knock downs of target genes and was based on the Na et al. 2013 publication. The sRNA is processed by the Hfq protein and then, can bind to the mRNA where it induces its degradation and stops the protein biosynthesis. Our system was designed according to the guidelines of the paper: a sRNA that binds with 24bp to the MinC mRNA (Figure X).

Some characteristics of minicells are – as the name suggests – the smaller size compared to their parent cells and the lack of chromosomal DNA. Thus, they are not able of further propagation.^[2] Apart from that, they contain all other parts of the parent cells.^[1]
Previous applications of minicells include drug delivery systems for the treatment of tumor cells to avoid inflammatory reactions which occur often when the drug is injected intravenously.^[3] Also, the usage in vaccines as antigen delivery systems were tested successfully.^[4] We did not find any evidence for toxicity or unwanted immunoreactions. This led us to the conclusion that the system could be FDA approved in the future.
To prevent infections as far as possible, minicells have to be isolated from their parent cells for these medical applications. Over the last decades, several methods were introduced. Chronologically, the first used technique was density gradient centrifugation.^[2] Because of limitations in sample sizes and low yields, antibiotic treatments of the minicell-producing strains were performed.^[5] Many antibiotics, e.g. Penicillin, kill bacteria during cell division, which is why they do not affect minicells. Lately, the treatment with antibiotics was combined with filtration to increase purification efficiency.^[1] In the future we will include aspects of those techniques in our purification methods to get less “normal” cells, when we are selecting for minicells.