Engineering Success of Kill Switch
Design
With respect to biosafety issues, we designed a kill switch system operating in engineered bacteria. In our design, MazE-MazF toxin-antitoxin with TetR gene, TetR repressible promoter, pBad promoter and RNA thermometer are used to control the survival of bacteria. We can switch on the killing system through temperature and chemical inducement. Here is our design:
Build
To check if the functions of each component in the design can regulate the expression of MazE as we expected, we built three constructs to evaluate the function. With Construct 2 & 3, the expression of MazE and TetR can be evaluated by testing the RFP intensity under different L-arabinose concentrations and temperatures. With the data collected from Construct 2 & 3, we can speculate the MazE expression in Construct 1.
The MazE and MAzF have been amplified by PCR from E. coli MG1655 genome DNA.
We successfully built our Construct 1 & 2 & 3. For more information, please visit our project Results page.
Test
To test how our kill switch design works, the expression of MazE and TetR can be evaluated by testing the RFP intensity under different L-arabinose concentrations and temperatures from Construct 2 & 3. We initially expected the bacteria with Construct 2 would show fluorescent red in LB medium (without L-arabinose). However, there was no red color in the colony of the bacteria, which means the RFP gene didn’t express. After using a photometer, the value of RFP intensity (RFP/OD600) is almost zero according to Fig.5, which is out of our expectation.
Learn
To figure out why the RFP didn’t express, we tested the function of the pBad promoter from Construct 3, which has a RFP gene regulated by pBad. The result shows that the leakage of pBad is huge according to Fig.7, which means that the TetR in Const 2 would still be expressed when there is no L-arabinose.
Then, we used modeling to analyze this system and we received the same answer. In an endogenous situation when there is no MazF in the bacteria, the expression of TetR wouldn’t be terminated, so the MazE won’t be expressed. Please see our project modeling for more information.
Re-Design
So we then try to construct the kill switch Design 2. In Design 2, we removed gene TetR and promoter pTetR and replaced them with a tandem promoter (a pBad with a constitutive promoter) which regulates the expression of MazF.
Here is our design:
Re-Build
To check if the function of our tandem promoter in the design can regulate the expression of MazF as we expected, we evaluated our design by testing the RFP intensity. However, we have to choose whether the promoter pBad or the constitutive promoter should be put in the former. Therefore, we built three constructs to evaluate the function. Strategy1 & 2 was designed to find where to put the pBad promoter, while Strategy3 was designed to compare more constitutive promoters in the same family of J23106.
We successfully used Gibson assembly to build Pre C1, Pre C2, and other tandem promoters (pJ102, pJ118, pJ110, pJ116, and pJ113). Please visit our Results page for more information.
Re-Test
The RFP expression of pBad located ahead (Pre C1) or behind (Pre C2) the constitutive promoter(J32106)
We found that Pre C2 is hardly induced by L-arabinose and the endogenous expression rate of Pre C2 is very low. However, the expression rate of Pre C1 is great when L-arabinose is added. In conclusion, the result showed that if we put pBad in the former, the expression of tandem promoter will be more stable. This experiment is conducted by our collaboration partner, 2021 TAS_Taipei. For more information about our collaboration, please visit our Collaborations page.
The RFP expression of Pre C1 compared with other 5 tandem promoters in the same family
We found out that 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 (BBa_J23102＞ BBa_J23118＞ BBa_J23106＞ BBa_J23110＞ BBa_J23116>BBa_J23113). 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.
Learn
First of all, we learned that tandem promoters with strong constitutive promoters like BBa_J23102, BBa_J23118 and BBa_J23110 have a better expression rate when there is no L-arabinose. After doing paper research [1], we found out that the mechanism of L-arabinose regulating pBad is that L-arabinose will bind and change the pBad inhibitor araC into an inducer. We suggest that the formation of L-arabinose-araC-pBad complex may be a huge barrier for RNA polymerase to transcript. Even though L-arabinose may enhance the expression of pBad, the interference of the transcription by the L-arabinose-araC-pBad complex plays an more important role.
Secondly, although the constitutive promoter J23106 in Pre C1 is from the same constitutive promoter family as others, they have quite different RFP expression characteristics. Besides, the strength of J23106 ranks only in the middle among others. The only difference is that Pre C1 is constructed in pSB1A2, while the others are constructed in pSB1C3. We suggest it is the difference of plasmid backbones that lead to different results. Hence, we are going to transfer these tandem promoters into pSB1A2 to see if these tandem promoters will have different results.
In conclusion, from Design 1 to Design 2, we should choose the promoter before MazE wisely. There are some pBad tandem promoters whose RFP expression ability will drop as the concentration of L-arabinose increases such as pJ102, pJ118, pJ110. 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. As the MazF surpasses MazE, the killing system will turn on and achieve the kill switch.
References
With respect to biosafety issues, we designed a kill switch system operating in engineered bacteria. In our design, MazE-MazF toxin-antitoxin with TetR gene, TetR repressible promoter, pBad promoter and RNA thermometer are used to control the survival of bacteria. We can switch on the killing system through temperature and chemical inducement. Here is our design:
Fig .1 Our kill switch design 1.
Build
To check if the functions of each component in the design can regulate the expression of MazE as we expected, we built three constructs to evaluate the function. With Construct 2 & 3, the expression of MazE and TetR can be evaluated by testing the RFP intensity under different L-arabinose concentrations and temperatures. With the data collected from Construct 2 & 3, we can speculate the MazE expression in Construct 1.
Fig. 2 Schematics of our biobrick construction
Fig. 3 Agarose electrophoresis in order to verify MazE and MazF genes. The purified DNA fragments had the expected length and were subsequently used for cloning.
Lane 1, 100bp ladder.
Lane 2, MazE PCR product.
Lane 3 and 4, MazF PCR product.
Land 5, 100bp ladder.
Fig .4 Agarose electrophoresis in order to verify Construct 1 and Construct 2.
Lane 1, 100bp ladder. Lane 1’, partial of Life 1 kb Plus ladder.
Lane 2 and 3, Construct 1 digested with BamHI.
Lane 4, Construct 2 digested with KpnI and NdeI.
Lane 1, 100bp ladder. Lane 1’, partial of Life 1 kb Plus ladder.
Lane 2 and 3, Construct 1 digested with BamHI.
Lane 4, Construct 2 digested with KpnI and NdeI.
Test
To test how our kill switch design works, the expression of MazE and TetR can be evaluated by testing the RFP intensity under different L-arabinose concentrations and temperatures from Construct 2 & 3. We initially expected the bacteria with Construct 2 would show fluorescent red in LB medium (without L-arabinose). However, there was no red color in the colony of the bacteria, which means the RFP gene didn’t express. After using a photometer, the value of RFP intensity (RFP/OD600) is almost zero according to Fig.5, which is out of our expectation.
Fig. 5 After 24 hrs 37 celsius degree cultivation, the RFP intensity of construct 2.
Fig. 6 The Construct 2 state in LB medium (without L-arabinose)
Learn
To figure out why the RFP didn’t express, we tested the function of the pBad promoter from Construct 3, which has a RFP gene regulated by pBad. The result shows that the leakage of pBad is huge according to Fig.7, which means that the TetR in Const 2 would still be expressed when there is no L-arabinose.
Fig. 7 The RFP intensity of construct 3(Pre B3) in different L-arabinose concentrations.
Fig. 8 Modeling result: the concentration of MazE according to time and the amount of MazF change.
Re-Design
So we then try to construct the kill switch Design 2. In Design 2, we removed gene TetR and promoter pTetR and replaced them with a tandem promoter (a pBad with a constitutive promoter) which regulates the expression of MazF.
Here is our design:
Fig. 9 Our kill switch design 2.
Re-Build
To check if the function of our tandem promoter in the design can regulate the expression of MazF as we expected, we evaluated our design by testing the RFP intensity. However, we have to choose whether the promoter pBad or the constitutive promoter should be put in the former. Therefore, we built three constructs to evaluate the function. Strategy1 & 2 was designed to find where to put the pBad promoter, while Strategy3 was designed to compare more constitutive promoters in the same family of J23106.
Fig. 10 Schematics of our biobrick construction
Fig. 11 Agarose electrophoresis in order to verify the tandem promoters.
Lane 1, 100bp ladder. Lane 2, pJ116 with BamHI and PstI. Lane 3, pJ118 with BamHI and PstI. Lane 4, pJ113 with BamHI and PstI. Lane 5, pJ110 with BamHI and PstI. Lane 6, pJ102 with BamHI and PstI.
Lane 1, 100bp ladder. Lane 2, pJ116 with BamHI and PstI. Lane 3, pJ118 with BamHI and PstI. Lane 4, pJ113 with BamHI and PstI. Lane 5, pJ110 with BamHI and PstI. Lane 6, pJ102 with BamHI and PstI.
Re-Test
The RFP expression of pBad located ahead (Pre C1) or behind (Pre C2) the constitutive promoter(J32106)
Fig. 12 The RFP intensity of Pre C1 and Pre C2 after 24 hr cultivation.
The RFP expression of Pre C1 compared with other 5 tandem promoters in the same family
Fig. 13 The RFP intensity of tandem promoters.
Learn
First of all, we learned that tandem promoters with strong constitutive promoters like BBa_J23102, BBa_J23118 and BBa_J23110 have a better expression rate when there is no L-arabinose. After doing paper research [1], we found out that the mechanism of L-arabinose regulating pBad is that L-arabinose will bind and change the pBad inhibitor araC into an inducer. We suggest that the formation of L-arabinose-araC-pBad complex may be a huge barrier for RNA polymerase to transcript. Even though L-arabinose may enhance the expression of pBad, the interference of the transcription by the L-arabinose-araC-pBad complex plays an more important role.
Secondly, although the constitutive promoter J23106 in Pre C1 is from the same constitutive promoter family as others, they have quite different RFP expression characteristics. Besides, the strength of J23106 ranks only in the middle among others. The only difference is that Pre C1 is constructed in pSB1A2, while the others are constructed in pSB1C3. We suggest it is the difference of plasmid backbones that lead to different results. Hence, we are going to transfer these tandem promoters into pSB1A2 to see if these tandem promoters will have different results.
In conclusion, from Design 1 to Design 2, we should choose the promoter before MazE wisely. There are some pBad tandem promoters whose RFP expression ability will drop as the concentration of L-arabinose increases such as pJ102, pJ118, pJ110. 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. As the MazF surpasses MazE, the killing system will turn on and achieve the kill switch.
Fig. 14 Our final kill switch design
References
[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.
