Revision as of 12:03, 10 October 2022

pDawn without LVA tag

Description

Our reduce the leakage level of pDawn system by removing the degradation tag LVA in the original repressor cI .

Sequence and Features

2022 SZPT-China

Biology

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.

Figure 1. Sequence alignment of pDawn(cI-LVA) before and after removing LVA tag

Usage

The plasmid containing the blue light induced kill switch was transformed into E. coli TOP10. Sixteen colonies were picked and cultured on two new LB plates at 37 °C for 16 hours. One plate was placed in the dark and the other was placed under blue light. The number of the candidates that survived the dark but failed to grow under blue light but survived in the dark was counted. For the E. coli strain containing pDawn(cI-LVA)-RBS070-LKD-pSEVA331, the number is 5. For pDawn(cI)-RBS070-LKD-pSEVA331, 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.

Figure 2. (a1) Colony growth of E. coli TOP10 containing the plasmid of pDawn(cI-LVA)-RBS070-LKD-pSEVA331 in the dark

(a2) Colony growth of E. coli TOP10 containing the plasmid of pDawn(cI-LVA)-RBS070-LKD-pSEVA331 under blue light

(b1) Colony growth of E. coli TOP10 containing the plasmid of pDawn(cI)-RBS070-LKD-pSEVA331 in the dark

(b2) Colony growth of E. coli TOP10 containing the plasmid of pDawn(cI)-RBS070-LKD-pSEVA331 under blue light

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-pSEVA331, 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-pSEVA331, 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.

Figure 3: (a) Colony growth of E. coli TOP10 containing the plasmid of pDawn(cI-LVA)-RBS070-LKD-pSEVA331 in the dark

(b) Colony growth of E. coli TOP10 containing the plasmid of pDawn(cI)-RBS070-LKD-pSEVA331 in the dark

From the above two groups of successful recombinant candidates (red squares in Figure 1), one colony was picked and grow in LB medium 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-pSEVA331 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-pSEVA331 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.

Figure 4: Growth curve of E. coli TOP10 containing pDawn(cI-LVA)-RBS070-LKD-pSEVA331

Growth curve of E. coli TOP10 containing pDawn(cI)-RBS070-LKD-pSEVA331