Difference between revisions of "Part:BBa K4632024"

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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).

pLas was from BBa_K2967001[1]

To achieve control over toxicant concentration, we introduced components labeled with BBa_K4632016 and BBa_K4632017 to create the T4-T4 lysis device. BBa_K4632016[2] and BBa_K4632017[3] was originally from BBa_K112805[4] and BBa K112806[5].

Sequence and Features

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 Escherichia 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 second set of verification systems 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 second set of engineering bacteria for verification system. There were 6 replicates per group.

Fig.3 Growth curve testing of the second set of verification system

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.