Part:BBa_K4632024
Quorum sensing-based T4 lysis device
Description
This part is a Quorum Sensing-based T4 lysis device constructed based on the LasI-LasR system and the T4-T4 lysis device.
The LasI-LasR system is a Quorum Sensing system from 'Pseudomonas aeruginosa.' As modified by Wei Jiang et al., it can be entirely orthogonal to the TraI-TraR system , another Quorum Sensing system from 'Agrobacterium tumefaciens' (Jiang et al., 2020).
1. How does it work?
In such a scenario, newly introduced engineered bacteria will promptly trigger the lysis gene, preventing the high-level expression of product expression., which could significantly reduce the accumulation rate of toxin product in the environment, creating a situation similar to a "threshold" for its accumulation.
2. What have we done? (SCAU-China 2023)
pLas was from BBa_K2967001[1] We introduced it to achieve control over drug concentration through bacterial density.
To achieve control over toxicant concentration, we introduced components labeled with BBa_K4632016 [2] and BBa_K112806[3] to create the T4-T4 lysis device. BBa_K4632016 was originally from BBa_K112805[4]. Its Codon was optimized for Escherichia coli expression.
The following gene circuit is designed as follows:(Figure 1)
Figure 1. Diagram of Quorum sensing-based T4 lysis device circuit design
We characterized the component to demonstrate its effectiveness, as detailed in the construction and characterization section.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 84
Illegal XbaI site found at 795
Illegal PstI site found at 367 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 367
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 84
Illegal XbaI site found at 795
Illegal PstI site found at 367 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 84
Illegal XbaI site found at 795
Illegal PstI site found at 367
Illegal AgeI site found at 1089
Illegal AgeI site found at 1159 - 1000COMPATIBLE WITH RFC[1000]
Construction and Characterization
In our initial validation experiments, we utilized a dual-plasmid system consisting of pBAD24M and pBAD33 to test our device. (Plasmid maps can be found in Figures 1)
1. Construction
First, both sets of plasmids were co-transformed into E. coli TOP10 using the KCM ice-cold method, and the success of transformation was confirmed using PCR.
Next, the successfully transformed engineered bacteria were streaked onto agar plates and incubated at 37 degrees Celsius. Single clones were selected and inoculated into liquid LB medium. Inducer was added, and the cultures were grown for 6 hours.
Fig.1 Diagram of the quorum sensing-based T4 lysis device circuit design
2. Validation of Product Expression Level Control
(1)Pre-experiment for Induced Expression of Lysis Effect
The successfully transformed engineered bacteria were streaked on plates and grown at 37°C. Single colonies were picked and inoculated into liquid LB medium, followed by the addition of inducers. The cultures were incubated for 6 hours.
Fig.2 Verification of lysis effect
The blank control was LB with 20% Ara. The control group consisted of the engineered bacteria without inducer, while the experimental group consisted of the engineered bacteria with a final concentration of 0.02% Ara. Each group had 3 replicates.
Result
After zeroing with the blank control, it was observed that the bacterial density in the experimental group decreased to 34.3% of that in the control group, indicating a significant lysis effect. This confirms the successful expression of quorum sensing and initiation of downstream lysis gene expression. Lysis gene expression was successful without leakage.
(2)Growth Curve Testing of the Second Verification System
Single clones were selected and inoculated into LB medium. After overnight incubation, the OD600 was adjusted to 0.6, and arabinose was added to a final concentration of 0.02%. The cultures were shaken for 21 hours in a sterile 96-well plate, and a growth curve was plotted. The blank control group was LB broth, the control group was wild Top10, and the experimental group was the engineering bacteria consisting of pBAD24M and pBAD33. There were 6 replicates per group.
Fig.3 Growth curve testing of the quorum sensing-based T4 lysis device
The growth curve revealed that the bacterial density continued to rise in the first 4 hours and began to decrease after 4 hours, stabilizing around the 9th hour. In contrast, the control group's bacterial density continued to rise.
At the 4th hour, the quorum sensing signal reached the threshold, initiating lysis gene expression. The engineered bacteria lysed, resulting in a significant decrease in bacterial density, which stabilized around the 9th hour.
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