Modeling
Hill function fitting
After the wet-lab tests were conducted, the florescence–concentration of ths/ttr was fitted using the formula:
F = Florescence
A = the fit of the minimum response with no thiosulfate
B = the maximum response at saturating thiosulfate concentration
k1/2 = concentration of thiosulfate at half-maximum response
n = Hill coefficient
Top10 and Nissle strains with the GFP system in aerobic and anaerobic environments
After the testing via Flow Cytometry, we obtained the following data for the ths sensor:
From these data, we can clearly see that the florescence of bacteria increased with increasing ths concentration.
We used the above-mentioned Hill equation to fit the data, and we found that the data can be fitted perfectly. The figure fitted using matlab is shown below: However, the data of bacteria with the ttr sensor were not satisfactory.
However, the data of bacteria with the ttr sensor were not satisfactory.
The bacteria were tested with only four inducer concentrations: 0mM, 0.01mM, 0.1mM, 1mM. Thus, we decided to do a second test of the GFP system.
Top10 and Nissle 1917 with the GFP system in an aerobic environment (more Data)
This time, we tested 7 inducer concentrations: 0mM, 0.01mM, 0.05mM, 0.1mM, 0.3mM, 0.7mM, and 1mM.
The data of this second measurement are shown below:
After data input into matlab, we obtained the figure shown below:
However, the ttr sensor still did not work as designed, so the data could not be fitted.
Consequently, we decided to test bacteria expressing the chromoproteins.
Coding
The code of modeling has been uploaded to GitHub:
https://github.com/AzirQuantum/SHSBNU_China_2017
Medicine Quantity
Bacteria Quantity
On November 16, 2011, Pharma Zentrale company sent an application to FDA and states that each their capsule contains E.coli strain 1917 corresponding to 2.5-25*109 viable cells.
The total weight of one cell is 9.5*10-13g.
Therefore, the weight of bacteria in one capsule is about 0.024g.
Color Testing
a. We cultured engineered bacteria overnight and then centrifuged them.
b. We added 1g curry, 500ul H2O and centrifuged bacteria.
c. We added 2g curry, 500ul H2O and centrifuged bacteria.
tube1&2 50ml LB+ TtrS/R- vio system in E.coli Top10
tube3&4 50ml LB+ ThsS/R- Pink system in E.coli Top10
Pink: spisPink vio:protoviolaceinic acid
Sample each of ttr-vio (ttrS/R sensor + protoviolaceinic acid biosynthesis pathway) and ths-pink (thsS/R sensor + spisPink chromoprotein) overnight cell culture for 100 uL and plate them on two different plates, we got:
Therefore, there are 5.2*109 bacteria in 20ml ttr-vio bacteria liquid, and its weight is about 0.050g.
There are 7.6*109 bacteria in 20ml ths-pink bacteria liquid, and its weight is about 0.072g, and 50ml is about 0.180g
Dose
We assumed 2.5×109 bacteria per capsule. The total weight of one cell is 9.5×10-13g. Therefore, the weight of bacteria in one capsule is about 0.024g.
References
Daeffler, K. N., Galley, J. D., Sheth, R. U., Ortiz‐Velez, L. C., Bibb, C. O., & Shroyer, N. F., et al. (2017). Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation. Molecular Systems Biology, 13(4), 923.
Frederick C. Neihardt (1996), Escherichia coli and Salmonella: Cellular and Molecular Biology (1st volume), ASM Press. Available at: http://kirschner.med.harvard.edu/files/bionumbers/Composition%20of%20an%20average%20E.%20coli%20Br%20cell-Neudhart.pdf
