Team:Marburg/Minicells
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
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 (crtY, 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 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
Proof of non-propagating minicell production
By flow cytometry and by fluorescence microscopy we can show that minicells are produced (Fig. 2,3). The minicells can be seen as a separated population in the side scatter that shows the complexity of the cells. Minicells have a lower complexity as they don’t contain any chromosomal DNA. To determine their shape and to get a better understanding of their phenotype, we additionally examined the minicells by scanning electron microscopy (Fig.4). Using strains with a fusion of HU, a histone-like protein, with the fluorescence protein GFP, we can show that the minicells indeed do not contain any chromosomal DNA (Fig.3). Performing microscopic time-laps experiments we can confirm on the one hand the expression of fluorescent proteins and on the other hand that minicells do not propagate anymore. This is a crucial aspect for the establishment of minicells as a tool with an application in the human gut.
TB43 HU-GFP. Additional proof of non propagating of the minicells
Purification
We were able to separate minicells from the parental strain with a purification of 98.8%. After non-successful efforts to separate the minicells by differential centrifugation, we developed our own purification method. We established a protocol with combination of different filter systems – first to exclude parental cells followed by a concentration process. Plating our concentrated cells indicates that there is only a slight background contamination with the parental cells. We will further improve this method in the future.
flow cytometry in the overnight culture and after the purification.
Production of nutrients
Induction of minicells
We can prove that the minicells can react to an inducer with the production of a fluorescent protein. By using an inducible promoter (pLAC) that controls a fluorescent protein (RFP), we could follow the production of RFP in time-laps fluorescent microscopy (Fig. 9). Our results show that non-fluorescent minicells became fluorescent after induction. This result demonstrates that minicells are can perform protein-biosynthesis and are able respond upon induction. Although most of the minicells only became fluorescent after induction, some cells constitutively expressed RFP, likely due to stochastic distribution of repressor molecules.
Modular device to transform to minicell producer
We were able to develop a tool for producing minicells out of any desired strain. For that a plasmid, which expresses the modular sRNA anti-minC, with a complementary sequence to the mRNA of minC, was constructed based on BBa_K1137009. NEB Turbo cells were successfully transformed with the sRNA plasmid(BBa_K1650011). Upon binding of the sRNA to the mRNA, the mRNA couldn’t be processed by ribosomes anymore, thus, leading to a knock down in MinC. Without MinC cell division is not located at midcell anymore and minicells are formed. The formation of minicells was confirmed by flow cytometry, where we used non-transformed cells grown to mid-log phase as a negative control (Fig.10)
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 the 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. Furthermore, 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 [Jivrajani et al. 2013]. 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.
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 [MacDiarmid et al. 2007]. Also, the usage in vaccines as antigen delivery systems were tested successfully [Carleton et al. 2013]. 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 [Adler et al. 1967]. Because of limitations in sample sizes and low yields, antibiotic treatments of the minicell-producing strains were performed [Levy 1970]. 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 [Jivrajani et al. 2013]. 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.
