ccdB cassette

All ccdb parts have been withdrawn from the Registry. Samples of parts containing the ccdB gene cannot be requested. - iGEM HQ

A positive selection marker useful for cloning. It includes a promoter and ccdB. ccdB encodes a protein toxic to most strains of Escherichia coli.

BBa_K4942010 Documentation

Improvement by Team 2023 SHSID-China

Group: SHSID-China iGEM 2023

Summary:

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 combining 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 expands the usage of ccdB and contributes to the biosafety career.

Construction Design and Engineering Principle

Some genetically modified microorganisms used in the production of engineered probiotics or industrial fermentation strains require special precautions for biosafety^[1]. 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 contamination^[2]. 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 microorganisms^[3]. 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 redesign ccdB by introducing it 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

Characterization/Measurement

We transformed the plasmid pTRIP-ccdB into E. coli DH5α and E. coli 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 3, 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. coli 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 3. Comparison of OD600 at 37°C and 22°C pTRIP-ccdB (E. coli 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. coli BL21 was the control group. According to Figure 4, 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 values. This test result is consistent with that for DH5α as discussed previously and this also backs up the engineering success of our temperature-based “kill switch,” the plasmid pTRIP-ccdB in E. coli host.

Figure 4. The Growth ability of pTRIP-ccdB in E. coli BL21 (DE3)

Reference

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
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
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. https://doi.org/10.1016/j.isci.2023.106165

Sequence and Features

Assembly Compatibility: