. Contribution .

Overview

As we embarked on our project, we read about the research and contributions of our predecessors. Their experiences provided us with a wealth of information, igniting many sparks of inspiration and insight. By integrating their established knowledge, we tailored our design to align closely with our chosen topic, propelling our project significantly forward.

We believe that enrich the collective knowledge base for future iGEM participants must be one of our goals. We expect that the contributions we made will inspire the other teams. All of this would realize one produces two and two produce more and make the iGEM community more colorful.

Contribution From XMU-China 2023

The ccdB gene is located on the Escherichia coli F-factor plasmid and it is a part of the toxin-antitoxin system encoded by the ccd operon. This part (BBa_K3512001) was first registered in 2020 (2020 BITSPilani-Goa_India) and used as a toxin in biosecurity applications.

For the contribution of this part, the survival assay was implemented to characterize the effect of the toxin, and the results were added to the page of corresponding part (1).

1.Construction of gene circuit

We used pBAD/araC (BBa_I0500), RBS(BBa_B0034), and ccdB (BBa_K3512001) to construct the composite part (BBa_K4907140) which were assembled on pSB4K5 backbone by standard assembly (Fig. 1).

This constructed circuit was transformed into E. coli DH10β, followed by positive transformant selection using kanamycin and confirmation through colony PCR (Fig. 2) and sequencing.

2. Characterization of killing effect

We used promoter BBa_I0500 to regulate the expression of CcdB. After induction by L-arabinose, OD₆₀₀ values were measured every two hours. The bacteria that had no CcdB expressed grew rapidly, while the one expressing CcdB showed a significant growth defect, as the optical density (at 600 nm) increased very slightly (Fig. 1a).

At each time, the spot assay was also performed, then the cell viability was measured by CFU count (Fig. 3b). Consistent with the trend of OD₆₀₀ value against time, only the absence of CcdB allowed the host cells to survive. All these results indicated that CcdB was toxic enough to the engineered bacteria so that this toxin could be applied to the cases when the suicide of genetically engineered microorganisms (GEMs) were strongly needed.

3. The neutralisation of CcdA to CcdB

ccdA is another gene in the ccd operon that encodes the anti-toxic protein CcdA, which protects cells from the toxic effect of CcdB via neutralisation. DB3.1 Super Competent Cell is a kind of E. coli which is capable of efficiently expanding the plasmid carrying bacterial toxic gene ccdB cause the ccdA is located on its genome. To validate the neutralization of ccdA to ccdB, we performed a series of dual-plasmid transformations to different E. coli strains as shown in Table. 1. The correct dual-plasmid system was confirmed by chloramphenicol and kanamycin.

The results showed that E. coli DB3.1 transformed with toxin genes and E. coli DH10β transformed with pBAD (BBa_K206001) promoter or pRha (BBa_K914003) promoter grew well. E. coli DH10β transformed with both toxins and antitoxins also exhibited good growth. However, E. coli DH10β with toxins did not grow. This confirmed the killing effect of the toxin CcdB again and the neutralization of antitoxin CcdA. Besides, the E. coli DB3.1 transformed with toxin controlled by pBAD promoter or pRha promoter and without antitoxin both grew better compared with E. coli DH10β. From these results, we can draw the conclusion that whether the ccdA is in plasmid or genome can play the role of neutralisation to ccdB.

Reference

1. C. T. Chan, J. W. Lee, D. E. Cameron, C. J. Bashor, J. J. Collins, 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat Chem Biol. 12, 82-86 (2016).