Overview
Synthetic biology is principally concerned with the rational design and engineering of biologically based parts, devices, or systems. As in any other engineering field, synthetic biologists follow iterations of the Design-Build-Test to successfully develop innovative biological systems (figure 1).
In the framework of our project, we first designed what parts we used, then assembled them into chassis, finally tested its effective. We through many times of iterations in order to construct a targeted therapy engineered microbe. Here we only describe in detail the process of multiple iterations of the pH promoter pCadC, about which the entire successful results of our successful construction of the engineered strain can visit proof of concept.
Cycle 1: Initial Testing of pH Promoter
Design
According to the literature, the pH-sensitive promoter pCadC, regulated by membrane-tethered activator protein (CadC), exhibits higher activity in acidic media than in neutral pH media (figure 2). So we chose this part to response to the low pH microenvironment around the colorectal tumor. The part is BBa_K4156076.
Build
So we added mRFP after the promoter and wanted to initially test the response of this promoter to low pH based on the fluorescence intensity. We use E.coli Nissle 1917 as chassis.
Test
We continuously measured the response of this promoter at different pH over 132 hours, characterized by normalized fluorescence intensity (figure 3). The results show a progressive enhancement of its response profile as the pH decreases. These could indicate that the pH promoter is effective.
Learn
But in the early stage, i.e. before 23 hours, we found that this promoter was very unstable in response to the signal, so we decided to add the amplification genetic switch. As for how to find this part, more details can be found by visiting integrated human practice.
Cycle 2: Make It More Stable
Design
In order to enable living cells to perform complex signal processing operations, amplifying genetic switches and Boolean logic gates based on serine integrase (Bxb1, TP901) are used in the design of biosensor systems [1]. These genetic devices enable bacteria to perform reliable detection, multiplex logic and data storage of clinical biomarkers in human clinical samples [2，3] to meet the requirements of medical testing.
To be specific, amplifying genetic logic gates use the asymmetric transcription terminator as the reversible switch to control the RNA Pol flow between gate input and output (figure 4). However, only when serine recombinase catalyze the unidirectional reversion of DNA in the corresponding recognition site can the status of signal output be changed.
Build
We added switch, which is TP901 and XOR gate, then followed with mRFP to characterize (figure 5). We also used E.coli Nissle 1917 as chassis organism.
Test
We continuously measured the response of this promoter, namely pH reporter, at different pH over 132 hours, characterized by normalized fluorescence intensity (figure 6). The fluorescence intensity of the pH reporter after adding switch appeared more stable over time at pH 7.3 and was higher than that of the reporter at pH 5.8, 6.3, and 7.3, which achieved the effect we wanted.
Learn
We initially achieved what we wanted - a biosensor that can steadily respond to low pH signals. Not only is this a solid foundation for our next iteration, but it shows that amplified genetic switch can indeed stabilize pH biosensors.
Cycle 3: Lysis!
Design
Because we have therapeutic proteins that cannot be exocytosed, it is not enough to simply stabilize the response signal, and we intend to add bacteriophage lysis gene phiX174E parts that will enable bacteria lysis.
Build
We added phiX174E to the above genetic parts (figure 7). We also used E.coli Nissle 1917 as chassis organism.
Test
We continuously measured the OD600 values of the engineered strains in the above four pH over 132 hours (figure 8). The lower $OD_{600}$ values indicate better lysis of the bacteria. The results showed that as the pH decreased, so did the $OD_{600}$, indicating that indeed the pH induced lysis of the strain and that phiX174E was effective.
Learn
After three iterations we have constructed a genetically engineered circuit that meets our expectations, and the next step is to select the appropriate chassis organism.
Cycle 4: Which is the Best Chassis?
Design
Based on the above validation, we can assume that strains were constructed that can stably respond to low pH. Since the chassis organism must be E.coli, but we started to think in which strain this gene circuit is responding better. So we wanted to compare it in E.coli Nissle 1917 and E.coli DH 5-alpha.
Build
The data were recorded at 2-hour intervals over 48 hours of induction at the same four pH values as before, and finally plotted as the normalized fluorescence intensity (figure 9). It can be observed that the circuit responds with higher intensity in E.coli Nissle 1917 than in E.coli DH5-alpha, so E.coli Nissle 1917 is a better chassis organism.
Test
The data were recorded at 2-hour intervals over 48 hours of induction at the same four pH values as before, and finally plotted as the normalized fluorescence intensity (figure 9). It can be observed that the circuit responds with higher intensity in E.coli Nissle 1917 than in E.coli DH5-alpha, so E.coli Nissle 1917 is a better chassis organism.
Learn
Since E.coli Nissle 1917 responded better as the chassis organism, we chose it as the chassis organism to construct our engineered bacteria in our subsequent experiments.
Conclusion
Through four iterations of the pCadC promoter, we have obtained a biosensor that can respond stably to low pH and can lyse the parent strain, which laid a solid foundation of our subsequent work.
Reference:
[1] Courbet A, Endy D, Renard E, Molina F, Bonnet J. Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates. Sci Transl Med. May 27 2015;7(289):289ra83. doi:10.1126/scitranslmed.aaa3601
[2] Benenson Y. Biomolecular computing systems: principles, progress and potential. Nat Rev Genet. Jun 12 2012;13(7):455-68. doi:10.1038/nrg3197
[3] Bonnet J, Yin P, Ortiz ME, Subsoontorn P, Endy D. Amplifying genetic logic gates. Science. May 3 2013;340(6132):599-603. doi:10.1126/science.1232758