The blue light response part pDawn is used to control the expression of lysis genes and characterized in the resultant system (Figure 1). The host chassis expressing this system is expected to grow normally without blue light and to lyse under blue light. However, we found that there is a significant proportion of the recombinant colonies did not grow very well in the dark, indicating a leak expression of the lysis genes (Figure 2-a). The colony which grew normally in the dark was picked and grown to logarithmic growth phase. But the strain continued growing under blue light, which means that the system became disabled in rapidly growing cells (Figure 2-b). It is suggestd that the above mentioned leaky expression of the lysis genes leads to loss-of-function mutation and that the favorable mutant overtakes the parental population.
Our goal is to reduce the leakage level of pDawn system by removing the degradation tag LVA in the original repressor cI as increasing the concentration of cI could enhance the repression of pR promoter.
The plasmid containing the blue light induced kill switch was transformed into E. coli TOP10. Sixteen colonies were picked and grown on two new LB plates at 37 °C for 16 h. One plate was placed in the dark and the other was placed under blue light. The number of the candidates that survived in the dark but failed to grow under blue light was counted. For the E. coli strain containing pDawn(cI-LVA)-RBS070-LKD, the number is 5. For pDawn(cI)-RBS070-LKD, the number is 10. The leakage expression of the lysis cassette LKD could be associated with this discrepancy in functionality. The new pDawn in which the LVA tag of cI is deleted improves the effectiveness of the blue light responsive lysis system.
From the above two groups of successful recombinant candidates (red squares in Figure 1), one colony was selected and streaked on a LB plate respectively. For each strain, sixteen colonies were picked and grown on two new LB plates at 37 °C in the dark. The growth condition of the bacteria on each plate was observed after 12-16 h. We found that for the strain containing pDawn(cI-LVA)-RBS070-LKD, a significant proportion of the recombinant colonies did not grow very well in the dark, indicating a leak expression of the lysis genes. Nevertheless, for the strain containing pDawn(cI)-RBS070-LKD, all the colonies grow normally in the dark. Therefore, the new pDawn in which the LVA tag of cI is deleted reduces the fitness cost of the blue light responsive lysis system, while the system with the original pDawn confers a significant growth disadvantage.
From the above two groups of successful recombinant candidates (red squares in Figure 1), one colony was picked and grown in LB media in the dark until OD600~0.6 was reached to track the growth condition under blue light. Then the OD600 values of the two selected strains under blue light was measured to compare the effectiveness of the two systems in rapidly growing cells. We found that both of the strain continued to grow during the first few hours of blue light illumination. However, only the strain containing the plasmid of pDawn(cI)-RBS070-LKD showed a significant decrease in OD600 value, indicating maintenance of lethality. And the OD600 value of the strain containing the plasmid of pDawn(cI)-RBS070-LKD increases a little bit, implying that loss-of-function mutations could occurred in these genes or in the host genome and that the favorable mutant overtakes the parental population. Therefore, the new pDawn in which the LVA tag of cI is deleted increases the evolutionary stability of the blue light sensitive kill switch by negating the deleterious evolutionary pressure of leaky toxin expression.
[1] Ohlendorf, R., Vidavski, R. R., Eldar, A., Moffat, K. & Möglich, A. From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. J. Mol. Biol. 416, 534-542 (2012).
[2] Ceyssens, P.-J. et al. Genomic analysis of Pseudomonas aeruginosa phages LKD16 and LKA1: establishment of the phiKMV subgroup within the T7 supergroup. J. Bacteriol. 188, 6924-31 (2006).
[3] Guiziou, S. et al. A part toolbox to tune genetic expression in Bacillus subtilis. Nucleic Acids Res. 44, 7495-7508 (2016).
[4] Stirling, F. et al. Rational Design of Evolutionarily Stable Microbial Kill Switches. Mol. Cell 68, 686-697.e3 (2017).
