An important aspect of "Natto it out" lies in ensuring that our recombinant live Biotherapeutic Product (LBP) will be under the restricted control of biosafety. In our biosafety project, MazE-MazF toxin-antitoxin genes serve as the conductor of the kill switch system. The Tandem promoter is the crucial component we utilize in the system. We construct the L-arabinose inducible tandem promoter before the MazF gene. In general, the level of MazE protein is higher than MazF; however, when the tandem promoter is induced by L-arabinose, the level of MazF will surpass that of MazE. To test the expression rate of tandem promoters and to find a suitable tandem promoter, we conduct the biosafety measurement.
We created seven kinds of tandem promoters constructed by many single promoters in iGEM kit.
Fig 1. Schematics of our biobrick construction
For constructing the tandem promoter, we have to choose whether the promoter pBad or the constitutive promoter should be put in the former. Hence, we constructed two kinds of tandem promoters with pBad. One is the J23106 ahead of pBad, and the other is the J23106 behind pBad.
Fig 2. The RFP intensity of Pre C1 and Pre C2 after 24 hrs cultivation
Pre C1: pBad-J23106 Pre C2: J23106-pBad
When testing the RFP result, we invited our collaboration partner, TAS iGEM team, for help in the measurement. We found that Pre C2 is hardly induced by L-arabinose and the endogenous expression rate of Pre C2 is very low. On the other hand, the expression rate of Pre C1 is great when L-arabinose is added. The result showed that if we put pBad in the former, the expression of tandem promoter will be more stable. For more information about our collaboration, please visit our collaboration page.

After finding out that putting pBad in the former is the better choice, we generated five other tandem promoters with Strategy 3 to construct them rapidly. In these 5 tandem promoters, we selected five other constitutive promoters (BBa_J23102, BBa_J23118, BBa_J23106, BBa_J23110, BBa_J23113) in the same family of J23106. (Promoter strength:BBa_J23102>BBa_J23118>BBa_J23106>BBa_J23110> BBa_J23116>BBa_J23113). We checked the strength of the tandem promoters by testing the RFP expression from each tandem promoter.
Fig 3. The RFP intensity of tandem promoters
According to the figures, when the concentration of L-arabinose is 0 M, the order of the expression rate of these tandem promoters is the same as that of the constitutive promoter individually. Besides, when the concentration of L-arabinose rises from 0 M to 0.025 M, the expression of RFP under the regulation of pJ102, pJ118, and pJ110 decreases while others rise. Interestingly, among these six tandem promoters, only the Pre C1 will be induced by L-arabinose significantly.
According to the result of the RFP expression of each tandem promoter, there are some pBad tandem promoters whose RFP expression ability will drop as the concentration of L-arabinose increases such as pJ102, pJ118, pJ110, which are all with strong constitutive promoters. Here we take pJ102 for example. We can construct pJ102 before MazE while Pre C1 before MazF. Under this design, in general, the expression of MazE will be higher than MazF, since when the concentration of L-arabinose is zero, the RFP expression of pJ102 is better than that of Pre C1. When the L-arabinose concentration increases, the expression rate of pJ102 will drop while that of Pre C1 will increase simultaneously.
Fig 4. Kill switch design according to the measurement
Take the 24 hrs L-arabinose concentration to RFP intensity for example. According to the line chart, we can see that there is a cross between Pre C1 and pJ112 between 0-0.025 M of L-arabinose. Assuming the change of RFP intensity according to L-arabinose concentration 0 to 0.025 M is fixed, we can get that the line of the L-arabinose concentration(x axial) to RFP intensity(y axial) might be:
Fig 5. The calculation of the ideal concentration of L-arabinose
The solution of these two function will be, while X stands for the concentration of L-arabinose
Fig 6. The calculation result of the ideal concentration of L-arabinose
From the above, we can turn on the killing system by taking enough of L-arabinose, approximately 6nM. Then, the expression of MazE under the control of pJ102 will drop and that of MazF under the control of Pre C1 will surpass the expression of MazE. As the MazF surpasses MazE, the kill switch system will turn on. The antitoxin MazE can not stop MazF from killing the bacteria.
[1] Öztürk, S., Ergün, B. G., & Çalık, P. (2017). Double promoter expression systems for recombinant protein production by industrial microorganisms. Applied microbiology and biotechnology, 101(20), 7459–7475.
Authored and maintained by Team NYCU-Taipei 2021.