New Improved Part: BBa_K4942010 (pTRIP-ccdB)
Existing Part: BBa _ P1011 (ccdB cassette, created by BE.109 Instructors)
BBa P1011 ccdB encodes a protein toxic to most strains of Escherichia coli. In our team, by combination of the ccdB protein expression and the temperature-based regulation engineering of pTRIP (BBa_K4942006), we can form a temperature-based “kill-switch”, pTRIP-ccdB (BBa_K4942010) thereby preventing the leakage of engineered microorganisms
Based on BBa _ P1011 (ccdB), we constructed a new combination plasmid BBa _ K4942010 (pTRIP-ccdB ) by attaching to the carrier BBa_K4942006 (pTRIP) which we created as a temperature control system. By combination of the ccdB protein expression and the temperature-based regulation engineering of pTRIP, we can form a temperature-based “kill-switch” thereby preventing the leakage of engineered microorganisms, which expand the usage of ccdB and make a contribution to the biosafety career.
Some genetically modified microorganisms used in the production of engineered probiotics or industrial fermentation strains require special precautions for biosafety1. It is important to prevent the unintentional release, multiplication, and spread of these genetically modified microorganisms into the environment, which could lead to unpredictable biological contamination2. This project has designed a simple and user-friendly "safety lock" for engineered microorganisms. Under normal conditions at 37℃ (the temperature inside the human body, which is also the working temperature for probiotics and commonly used in industrial microbial fermentation), the "safety lock" remains inactive, allowing the host microorganism to reproduce and function normally. However, at 22℃ (a temperature closer to natural environmental conditions, excluding tropical regions and extremely hot summers), the "safety lock" becomes active, expressing a toxic protein that leads to the self-destruction of the host microorganism, thereby preventing the release of the engineered microorganisms3. We constructed a temperature control system（pTRIP）, and we developed a plasmid, pTRIP-ccdB, which contains a toxic protein, (ccdB), that can kill bacteria upon expression, thereby preventing the leakage of engineered microorganisms(Figure 1).
Figure 1. The engineering design schematic diagram.
The ccdB is a toxic protein that needs to be transformed into E.coli DB3.1 competent cells. E.coli DB3.1 competent cells are anti-toxic. In this cycle, we will redesig ccdB by introduced into pTRIP to upgrade the toxic protein to a “kill-switch”(Figure 2). The recombinant plasmid pTRIP-ccdB was obtained by homologous recombination.
Figure 2 . The plasmid map of pTRIP-ccdB
We constructed the pTRIP-ccdB plasmid using homologous recombination. The PCR amplification of the ccdB sequence resulted in a fragment of 306bp in length. The figure 3 indicates that the amplified band matches the expected size, confirming the successful amplification of the ccdB sequence from the linearized plasmid.
Figure 3. The gel electrophoresis validation of ccdB.
By using the pTRIP plasmid as a template, we performed PCR amplification to obtain a 5473kb fragment referred to as pTRIP-C. The gel electrophoresis image in Figure 4 shows that the amplified band matches the expected size, indicating the successful amplification of the linearized pTRIP-C plasmid.
Figure 4. The gel electrophoresis validation of pTRIP-C.
The pTRIP-ccdB plasmid was transformed into E. coli DB3.1. The single clone colony growth on plates is shown in Figure 5 A and B. Clones 1-8 were selected for antibody verification, and the results in Figure 5C demonstrate clear bands, confirming the presence of the ccdB sequence with a length of 306bp. The gel electrophoresis image in Figure 4C matches the expected band, indicating the successful transformation.
Next, colonies 1-8 were sent for sequencing, and the sequencing results in Figure 5D showed a 100% match with the ccdB nucleotide sequence. This confirms the successful integration of the ccdB fragment into the pTRIP-E plasmid. It further validates the successful construction of the pTRIP-ccdB plasmid.
Figure 5. The monoclonal antibody validation and sequencing of pTRIP-ccdB (E.Coil DB3.1) .
A. Monoclonal plate for pTRIP-ccdB ( E.Coil DB3.1 )
B. Monoclonal verification gel of pTRIP-ccdB ( E.Coil DB3.1 )
C. Sequencing results of pTRIP-ccdB
Protein expression
The size of the ccdB protein, the target protein, is 11.7 kDa. Protein expression was induced at a concentration of 0.6mmol AI, and induction was performed at 37 oC and 22 oC. According to the figure 6, in the control group (line 1 -line 2 and line 9 -line 10), ccdB protein was not observed. Under the condition of 37 oC (line 3 to line 4), ccdB protein was not observed. However, under the condition of 22 oC (line 5 to line 8), a weak presence of ccdB protein is detected. This indicates that ccdB expression does not occur at 37 oC, while there is limited expression at 22 oC. Therefore, it can be concluded that our temperature control system is in the activated state at 22 oC and in the deactivated state at 37 oC.
Figure 6 : The SDS-PAGE of ccdB protein
Note:
1-2: E.coil DB3.1(control)
3-4：37oC-pTRIP-ccdB(E.coil DB3.1)
5-8：22oC-pTRIP-ccdB(E.coil DB3.1)
9-10: E.coil DB3.1(control )
We transformed the plasmid pTRIP-ccdB into E.Coil DH5α and E.Coil BL21 (DE3). Then ,the growth ability of pTRIP-ccdB at different temperatures of 22 °C and 37 °C by AHL induction was studied and validated and it also validated the sensitivity of our temperature-based “kill-switch”.
According to Figure 7, in two groups with AI (AutoInducers, herein it refers to N-(3-oxohexanoyl)-L-homoserine lactone) concentration of 0.6 mmol, the OD600 of pTRIP-ccdB at 37 °C increased firstly and then tended to be stabilized over time, while there was almost no significant change of that at 22 °C. It is seen that pTRIP-ccdB (E.coil DH5α) grew much better at 37 °C than 22 °C where it indicated little growth over time. This supports the conclusion that the bacterial strain grows normally at 37°C, while the presence of ccdB at 22°C leads to bacterial cell death.
.
Figure 7. Comparison of OD600 at 37°C and 22°C pTRIP-ccdB (E.coil DH5α ) with AI concentration of 0.6 at different times
Comparisons of growth capabilities were made at 37°C and 22°C with an AI concentration of 0.6 mmol.The E.coil BL21 was the control group. According to Figure 8, it is evident that the OD600 of BL21 (the control group) is significantly higher than that of BL21(pTRIP-ccdB) at 22°C while the OD600 of BL21 and BL21(pTRIP-ccdB) have similar value. This test result is consistent with that for DH5α as discussed previously and this also back up the engineering success of our temperature-based “kill switch”, the plasmid pTRIP-ccdB in E. coli host.
Figure 8. The Growth ability of pTRIP-ccdB in E.coil BL21 ( DE3 )
[1] Bazhenov, S.V., Scheglova, E.S., Utkina, A.A. et al. New temperature-switchable acyl homoserine lactone-regulated expression vector. Appl Microbiol Biotechnol 107, 807–818 (2023). https://doi.org/10.1007/s00253-022-12341-y
[2] Nocadello, S., Swennen, E.F. The new pLAI (lux regulon based auto-inducible) expression system for recombinant protein production in Escherichia coli. Microb Cell Fact 11, 3 (2012). https://doi.org/10.1186/1475-2859-11-3
[3] Hoffmann SA, Diggans J, Densmore D, Dai J, Knight T, Leproust E, Boeke JD, Wheeler N, Cai Y. Safety by design: Biosafety and biosecurity in the age of synthetic genomics. iScience. 2023 Feb 10;26(3):106165. doi: 10.1016/j.isci.2023.106165.