Part:BBa_K4632012:Experience

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Applications of BBa_K4632012

Experimental Results

(1)pnirB promoter characterization

The pnirB-Gm fragment was cloned to pBAD24M through digestion and ligation. Three rounds of PCR amplification were employed to introduce the pnirB promoter and restriction sites in front of the gentamicin (Gm) resistance gene. Successful transformation of this construct into bacterial strains was confirmed through Gm resistance selection. The colonies that could grow on the Amp+Gm plate were further checked by colony PCR (Figure. 2). Subsequently, the expression strength of pnirB were assayed under aerobic or anaerobic conditions with a gradient concentration of Gm.

Figure 2. Colony PCR of pBAD24M-pnirB-Gm resistant Lane1-3, PCR of pBAD24M-pnirB-Gm resistant

The successfully transformed strains were streaked onto eight different Gm concentration plates, ranging from 0/0.5/1/2/5/10/15/30 μg/mL. These plates were incubated for 48 hours under aerobic or anaerobic conditions (anaerobic conditions were achieved by placing the plates inside an anaerobic chamber).(Figure. 3)

Figure 3. Growth of E. coli Top 10 containing anaerobic promoter and Gm-resistant under different conditions

Under non-resistant and anaerobic conditions, bacterial growth is expected to be less favorable compared to aerobic conditions. As observed from the results, up to 2 μg/mL resistance concentration, there was no difference in bacterial growth between aerobic and anaerobic environments. Starting from 5 μg/mL, , only a few colonies were present in the initial streaked area under the aerobic environment. In contrast,, bacterial growth was still extensive even at 10 μg/mL under anaerobic conditions. This suggests that pnirB is specifically expressed under anaerobic conditions. However, when the Gm increased to 15 μg/mL, strain could not grow well neither under aerobic nor anaerobic conditions.

In conclusion, the expression of pnirB under anaerobic condition is at least two times higher than the aerobic conditions. This could help us to construct the Biological Safety Device.

(2) Biological safety device efficacy testing

E. coli wm3064 is a dapA mutant strain with diaminopimelic acid (DAP) nutritional defect. The dapA gene were ligated to the downstream of pnirB and cloned to pUC18T-mini-Tn7T plasmid, then transformed to wm3064. The growth of ΔdapA/dapA- pUC18T-mini-Tn7T and ΔdapA strains were tested under aerobic or anaerobic conditions. (Figure. 4)The results showed that ΔdapA could only grow in the presence of DAP no matter in aerobic or anaerobic conditions, while ΔdapA/dapA- pUC18T-mini-Tn7T could grow under anaerobic conditions without the supplementation for DAP.

These results(Figure. 4) indicate that this strain can only grow under anaerobic conditions and possesses biological safety. During production, growth can be ensured by adding DAP.

Figure. 4 Results of culture experiment of dapA gene defective strain<p> <p>This work qualitatively confirms that the pnirB element can indeed exhibit specific expression under anaerobic conditions. Furthermore, by utilizing pnirB to drive the essential diaminopimelic acid gene dapA, it restricts bacterial growth to anaerobic environments, such as the gut of the S. invicta. These findings provide a theoretical foundation for the next steps in the development of the biological safety device, specifically the recombination of pnirB into the E. coli chromosome.

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