Demonstration
Introduction
Our human practice included an online-and-offline survey, asking the public about their general information, experience and knowledge on gut inflammation, and their thoughts about using probiotics to detect and treat gut inflammation. We found that people generally are concerned about the following aspects: the product’s safety, harm to human body, sensibility, price, and response time. Thus, we decided to demonstrate our way of dealing with these problems and how can we meet the public’s need.
Safety
Safety is always the first concern that comes when a new product is posed; but our bacteria is generally satisfactory in safety.
First, E. coli Nissle 1917, the kind of bacteria that patients will take, is generally considered safe and even one of the best investigated therapeutically applied E. coli strain worldwide. E. coli Nissle 1917 is non-pathogenic, which means that it does not cause any disease or produce any toxins. E. coli strain Nissle 1917 does not possess any antibiotic resistance plasmids and is very stable, with a poor recipient of foreign DNA (especially those in the form of antibiotic resistance and other promiscuous plasmids). Despite the safe properties of Nissle 1917, we are still planning to do some engineering on its cryptic plasmids (Fred A. Hines et al., 2011) to make it produce appropriate amount of DNase at appropriate time, so that for those engineered bacteria that are left in the gut or excreted to the environment, it can automatically destroy its DNA when a specific time is reached or environment is present.
Second, we are planning to put the bacteria in gastro-resistant hard capsules to make sure that it does not emit to the environment. This capsule is able to permit the transmission of small chemical compounds, and we are now able to control when (or in what pH) the capsule would be dissolved. (Fred A. Hines et al., 2011)
Thirdly, one gastro-resistant hard capsule contains about 2.5 – 25×109 viable E.coli Nissle 1917 cells, which is within the safe range for human body proved by FDA. (Fred A. Hines et al., 2011)
Fourthly, violacein and chromo-protein generally have no toxicity or risk. Based on experiments done in vivo with violacein, daily intake of up to 1mg/kg for 35 days is well tolerated and doesn’t cause hematotoxicity nor pose risks of renal or heptatotoxicity (Bromberg et al., 2010). And chromo-protein is generally safe because it is all working with probiotic strain and making these strains to produce healthy nutritional compounds, and chromo-protein does not produce any kind of toxic or dangerous gene in any way (Hampus Elofsson et al, 2013).
Sensitivity
Our engineered bacteria show distinct color-depth difference when exposed to different concentrations of tetrasulfate and thiosulfate, ranging from 0.01 mM to 1mM. As it is tested in dextran sodium sulfate (DSS) mouse model, the two-sensor system was able to sense and report the tetrathionate and thiosulfate concentration level by expressing sfGFP; this is an indirect prove that our system is theoretically able to report different concentrations of tetrathionate and thiosulfate. Detail speaking, according to the diagrams found (shown as follows) in the passage “Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation,” the average fluorescence when DSS is present is about 2000 MEFL; relating this data to another diagram, 2000 MEFL of fluorescence corresponds to a thiosulfate concentration of about 0.1 mM, which can be considered an estimated average of the concentration of thiosulfate in human’s gut. Since the 0.1 mM concentration is within the range that we can visualize the difference of resulting color, we can say that our project have an acceptable sensibility (Kristina N-M Daeffler et al., 2017).
As we can see in the following photo, the expression of protoviolaceinic acid (a fore body of violacein) in different concentrations can be easily visually distinguished, so that its sensitivity can be guaranteed.
Price and Response Time
Our bacteria would be already modified and copied to an appreciable quantity when it is ready to be commercialized, so that the job for each seller would be just cultivating the bacteria, making the capsules, and transporting them within 2-8℃. Generally speaking, this whole process wouldn’t be very expensive compared the current ways of gut inflammation testing. We used the price of Mutaflor as a model and comparison; Mutaflor sells 20 capsules for $28.95, but our patients would only need one capsule for inflammation testing, so that the prime cost would be around $1.5 per capsule. This, is much cheaper than any methods currently commercialized (estimated prices of existing detection methods are listed below).
Blood testing (without insurance) would range from $700 to $2589; stool testing would generally cost $5 - $100, testing in hospitals are generally more expansive, and in most hospitals stool testing can only detect a few common compounds or bacteria (e.g. shigella and salmonella in China); colonoscopy is even more expensive, ranging $1000 - $3764.
Thus, our product will be able to offer a much more accessible price.
Then about the response time. As we did in our previous experiments, our bacteria can correctly and distinctly response to different concentration of the two chemical compounds after 12 hours of cultivation, which is shorter than the normal digesting time (14-24 hours). Considering the capsule (or the bacteria) might not get into contact with inflammation too early, so that there is still possibilities that the bacteria still haven’t have enough time to produce violacein or chromo-protein when digested. Since we might commercially start with insoluble capsules, patients might need to keep the capsule for 12 more hours to cultivate the bacteria and to let them show their color. When we further develop it into gastro-resistant hard capsule, some bacteria would be left in the gut for days, so that patients would be able to determine the inflammation level by observing the excrete of the next day.
For further instructions and details in using this capsule, see our guidance(Applied Design).
