Proof of Concept

1. Synthesized FadL and FadD function

We examined the function of the bioengineered product FadL and FadD. We added Palmitic acid—a long chain fatty acid frequently contained in fast foods— into standard LB liquid medium with engineered bacteria. The bacteria culture was shake-nurtured for 24 hours under 220rmp and 37 degrees Celsius. Then, we performed Differential Centrifugation to the sample, applying 1000rpm for 1 minute to isolate and remove unreacted Palmitic acid in the supernatant, then 3000rpm for 10 minutes to collect bacteria. Supernatant was then diluted with distilled water. The concentration of Palmitic acid in the supernatant and in the bacteria are measured using photospectrometer.

The testing was repeated for 3 times to ensure credibility. The result is as shown:

	Negative Control	Only FadL	Only FadD	FadL and FadD
Extracellular	++++++++	+++++	+++++++	+++
Intracellular	++	+++	+	++
Total	++++++++++	++++++++	++++++++	+++++

The total concentration of Palmitic acid the treatment group with both FadL and FadD genes is significantly lower than that of Negative control, which contain only the plasmid carrier but neither of the two genes, indicating that the LCFA intake and metabolization are achieved. Meanwhile, the treatment group with only FadL gene has a lower extracellular concentration and higher intracellular concentration of Palmitic acid than does negative control, indicating successful LCFA transportation.  The treatment group with only FadD gene, on the other hand, has a lower intracellular concentration of Palmitic acid than does negative control, indicating successful LCFA metabolism. Taken as a whole, the system can achieve the goal of Palmitic acid intake and metabolization in vitro, and both synthesized proteins involved in the process, FadL and FadD, are functional and effective.

2. Optimum pH and temperature

Secondly, we measured the optimum pH and temperature of our engineered bacteria to ensure their survival and appropriate functioning inside human intestine. We nurtured bacteria in pH 5~9 and temperature 25~50 degree Celsius and measured the Palmitic acid concentration in bacteria cultures using similar approach like in step one. Bacteria are treated under different pH and temperature and shake-nurtured in LB culture for 60 hours under 220rpm. Within each system, the initial Palmatic acid concentration is set at 20g/L. The consumption of Palmitic acid is then measured using the same approach in step one: centrifugating the bacteria liquid culture first at 1000rpm for 1 minute to separate Palmitic acid remaining in the culture and at 3000rpm for 10 minutes to collect bacteria, and then use photospectrometer to measure palmitic concentration in and out of the cells. The total amount of remaining Palmitic acid is deducted from the initial concentration to get total Palmitic acid consumption, which indicates the functioning of bacteria under different environment settings. The results are represented in the figures:

3. Bacteria Function and Survival at Simulated Human Intestinal Environment

Finally, we tested the function engineered bacteria in a simulated intestinal environment. To simulate the environment of intestine, the pH is controlled at 7 and the temperature is controlled at 37 degrees Celsius. Moreover, the system is kept anaerobic to simulate the scarcity of oxygen inside human intestine. To achieve this, the Erlenmeyer bottle that contains the experimental system will be put inside an anaerobic bag. Bile salt, Pancreatic juice, and lipase (purchased as bottles of powders, not taken from any animal or human) will be added to the bottle to simulate real contents of human intestine. Considered the main source of lipids in human body, 24 degrees Celsius Palm oil is dissolved into LB medium at temperature above 24 degrees Celsius. The experimental system that contains LB medium, Palm oil, Bile salt, Pancreatic juice, and lipase was shake-nurtured for 60 hours under 300 rpm. Palm oil consumption is measured after cooling the sample and weighing the gained solid. Palmitic acid consumption is measured using the same centrifugation approach as in the step of measuring the optimum pH and temperature.  After centrifuging and diluting, OD value at 600 nm can be measured to determine the relative concentration of bacteria bodies in the system. The result of Palmitic acid consumption is represented in the figure:

Both the test and the controlled bacteria can survive under simulated intestinal environment. However, Palmitic acid consumption of engineered bacteria that carries both genes, as indicated by the blue line, is almost twice of that of controlled bacteria that contains only the plasmid, as indicated by the red line. However, the engineered bacteria performs slightly less effective than it would have at optimum environment. 

4. Biosafety Double Kill Switch function

Finally, we conducted experiments to proof that the deprivation of L-Rhamnose activates the proposed Double Kill Switch system and induce cell death. E. coli with Kill Switch genes and without kill switch genes are nurtured in LB liquid culture respectively for 24 hours. From the beginning of the culturing, L-Rhamnose is added to both cultures. The culturing began at when both groups’ OD 600 equals 0.1. OD 600 is measured to indicate the concentration of bacteria body within the culture. At the 9th hour of culturing, L-Rhamnose is no longer added, and Rhamnase is added. The concentrations of control and test bacteria are compared. The result is represented in the graph: 

The result shows that the bacteria with kill switch, after a lagging time for around 3 hours after rhamnose is no longer added and rhamnase is added, began to significantly decrease in concentration, whereas the control group that carry no kill switch genes were unaffected by the deprivation of rhamnose and continue to increase in concentration. The comparison of the bacteria’s performance successfully demonstrates that the kill switch effectively kills the bacteria when L-rhamnose is not present in the environment.

5. Conclusion

We have proved that our project will likely work in a relevant context. We have demonstrated that our engineered bacteria synthesized functional FadL and FadD proteins, survive and function under human intestinal environments, and that our proposed kill switch can prevent possible escape of engineered bacteria to unintended environment.