Escherichia coli Nissle 1917 (EcN) is probably the most intensively investigated bacterial strain today[1]. Despite the fact that the EcN strain is widely used as a probiotic, a lot of questions remain. That is why we decided to dive deep into the characterization of EcN. Our goal was to find out more about EcN itself and to provide crucial information in order to include EcN as platform organism for other iGEM Teams and researchers.

The first step in the characterization of a bacterium are growth curves on different media and under different conditions. We chose to perform half of our experiments under aerobic and the other half under anaerobic conditions. Under aerobic conditions, as a control we let EcN grow in LB medium at 37°C and pH7. We changed the temperature to 25°C, 8°C, 4°C and 0°C, showing that EcN growth is inhibited at 8°C, the temperature often used for cold shock.

Moreover, we evaluated the growth in the pH range from pH6 to pH1, since in our application EcN will have to pass the acidity of the stomach. Here, at pH4 we discovered an interesting recovery of EcN growth after three hours.

To look into inflammatory stress, we subjected EcN to up to 100µM hydrogen peroxide [2] and showed that the growth is not substantially influenced by it.

Lastly, we performed dryfreezing and recovered EcN afterwards under control conditions [3][4][5], showing that dryfreezing within skim milk protects the bacteria from substantial death.

Anaerobic conditions were used to characterize EcN growth under the circumstances provided in the human intestines. Thus, as a control we compared EcN growth in LB medium versus mGAM medium under anaerobic conditions, showing that mGAM, a medium designed for anaerobic application, is in fact a more suitable medium. Then, we subjected the bacteria to metformin treatment, since it is one of the most common treatments of diabetes and is known to accumulate to 720 µM to 7,2 mM within the intestines due to low bioavailability. [6][7]. We showed that EcN growth is not substantially influenced by 1,3 mM of metformin into mGAM medium. Moreover, we tested for EcN’s resistance against cholic acid, since it is commonly secreted into the ileum. Here, we added 0.25 mM of cholic acid to mGAM medium, considering that the concentration of free bile salts may rise to 0.25 to 1 mM of total bile salts in the Ileum, with cholic acid only being one component [8][9]. Our results suggest that EcN growth is not greatly influenced by cholic acid.

Finally, we tested EcN interaction with other bacteria by adding bacterial supernatant to our medium. The supernatant was sterilized and provided by Dr. Lisa Maier. We chose Bacteroides thetaiotaomicron, Prevotella copri and Ruminococcus gnavus since they are commonly found within the human microbiome [10]. Bifidobacterium adolescentis was chosen due to its probiotic nature and Clostridium difficile supernatant was used, since it is often found in the microbiome of people with chronic inflammation [10][11]. The results suggest that EcN grows under all additions, however was initially inhibited in its growth by the addition of Bacteroides spp., thus we used these samples for RNA-seq.

The transcriptome describes the set of whole RNA molecules in a population of cells and is subject to continuous changes. Understanding the complete transcriptome, the expressed genes, post-transcriptional modifications, single-nucleotide polymorphisms (SNPs) and additional properties of interest is imperative towards understanding genetic cause, disease and possible treatment strategies. The inherent complexity of the transcriptome requires precise and scaling analysis techniques. RNA sequencing (RNA-seq), also known as whole transcriptome shotgun sequencing (WTSS), is most commonly used for this purpose, today. It uses next-generation sequencing (NGS) to detect and quantify RNA in biological samples. The generated RNA-Seq read data is then analyzed according to a sample RNA-Seq work-flow as shown below.

We set ourselves the goal of understanding the transcriptomic changes that E.coli Nissle undergoes under various stress conditions to gain a deeper insight into its responses. Understanding the stress responses of E.coli Nissle could lead to the development of more stress robust strains, which not only our project would benefit from, but also scientists working on probiotic drugs in general.

We divided our experimental design into two parts. We not only wanted to investigate the effect of environmental stress on E.coli Nissle under aerobic conditions, but were also specifically interested in anaerobic conditions. Hence, our experimental design looks as follows:

The respective temperature, pH values and doses were determined by growth curves, shown above. To get the most out of RNA-Seq applied to stress factors, it is important to find the cutoff values, where E.coli Nissle is put under stress the most, but still survives.

Sequencing and library preparation was conducted on two sites. All aerobic samples were prepared and sequenced at the NGS Competence Center Tübingen (NCCT), whereas all anaerobic samples were prepared and sequenced at the European Molecular Biology Laboratory (EMBL) in Heidelberg. For more details please visit our Notebook and Attributions.

Quality Control Differential Expression Analysis Pathway Analysis

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