Part:BBa_K4849023:Design
E. coli NucA/NuiA kill switch
Design and Experimental confirmation
The assembled LvT E. coli Kill Switch construct was transformed into competent Escherichia coli TOP10 cells. Colony PCR was used to screen for colonies with the correct insert size. The expected amplicon size was 1930 bp. Colonies 2, 3, and 8 gave the expected amplicon size (Figure 5).
Figure 5. Colony PCR of Level T E. coli Kill Switch construct.
We set up two overnight cultures, one from colony 2 that gave the correct cPCR band size for level T E. coli KS construct, and the other one from a blue colony containing the empty vector (WT+vector). We used these initial cultures to make new cultures in triplicates with or without added 400 µM Zn2+ for both KS and WT+vector control. For KS characterisation, we grew these cultures in a shaking incubator at 37C, 160 rpm, and took OD600 measurements at 1 hour intervals to produce growth curves (Figure 6). From growth curves, cells containing Kill Switch plasmid or empty vector grew better without added zinc, indicating that the addition of 400 μM ZnSO4 slows down cell growth. Importantly, there was no apparent decrease in growth of cells containing the Kill Switch in media without added zinc compared to those grown with added zinc. This could be due to nuclease not being expressed, thus unable to kill the cells. Notably, NucA requires divalent metal ions like Mn2+ or Mg2+ as cofactors, the optimal concentration for these being around 5 mM, while NucA activity was shown to decrease with increasing concentration of monovalent salt. LB media contains a high concentration of NaCl but no divalent salts, meaning that NucA might not function due to lack of cofactors. Alternatively, zinc-inducible promoter might not function as expected, leading to constitutive expression of antitoxin, thus inhibiting nuclease. In future experiments, NucA and NuiA expression should be verified by Western Blotting before characterizing the Kill Switch. Also, LB media should be supplemented with appropriate concentrations of divalent salts to ensure the presence of cofactors for NucA.
Figure 6. Growth curves of E. coli TOP10 cells with or without Kill Switch plasmid in media with or without added 400 μM ZnSO4.
For checking the viability of cells containing the KS in conditions with and without added zinc compared to WT+vector control we performed colony forming unit (CFU) counting assay (Figure 7). In general, the experiment was unsuccessful since the numbers of colonies observed after plating 10 µL of culture diluted by the factor of 107 were too low to be able to accurately deduce the CFU number in original undiluted samples. Thus, in the future the experiment should be redone by plating lower dilution or higher volume of cells. Moreover, as noted previously, since NucA requires divalent metal ions like Mn2+ or Mg2+ as cofactors, ideally the plates should be supplemented with them. From our results, at 6 hours, no significant difference in CFU/mL numbers was observed between KS-containing cells grown with or without added zinc (2 sample t-test, p-value > 0.5).
Figure 7. CFU/mL (log10) values for E. coli TOP10 cells with or without Kill Switch plasmid in media with or without added 400 μM ZnSO4. KS and WT+vector cultures (in triplicates) were grown in media with or without zinc were sampled every 2 hours (for 6 hours total), diluted, and plated onto LB + Spec + 400 µM Zn2+ and LB + Spec (without zinc) plates, respectively.
Source
PzntA-mutant synthesised zinc-inducible E.coli promoter; NucA-from Anabaena sp. PCC 7120 but we modified and synthesised; BBa_J23114 Anderson promoter; NuiA - from Anabaena sp. PCC 7120 but we synthesised; TECK terminator - E. coli; TrrnB-synthetic
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