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| − | + | Fig1: Schematic of test quorum sensing device (ptetR-LasR-pluxR-GFP). Promoter luxR was activated by LasR-3OC12HSL complex, leading to produce GFP. | |
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| + | Fig2: Characterization results of quorum sensing device. a| GFP production rate per cell over time at different concentration of 3OC12HSL. b| Time-average GFP production rate per cell over time at different concentration of 3OC12HSL. | ||
===User Reviews=== | ===User Reviews=== | ||
Latest revision as of 06:30, 8 October 2023
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Applications of BBa_K4785003
The sensing device was designed based on the Type I quorum sensing mechanism of P.aeruginosa to effectively produce PslG and HMGB1 only in response to the presence of P.aeruginosa (Fig 1a). The tetR promoter constitutively enhances expression of transcriptional factor LasR, which can bind to AHL 3OC12HSL. Then, LasR-3OC12HSL activator complex can open luxR promoter, leading to production target proteins.
To characterize the sensing device, the gene encoding the green fluorescent protein (GFP) was assembled to the device (Fig 1b). Through measuring GFP synthesis rate, we can simulate expression of target protein under a range of concentration of 3OC12HSL (Fig 2a). We observed a basal expression level of 0.427 RFU per OD per minute with lower concentration (< 5.0E-9M) of 3OC12HSL induction, followed by a sharp increase in GFP production rate as the concentration of 3OC12HSL was increased beyond 1.0E-8M. GFP expression achieved a peak at 1.0E-7M. These results announced that the optimal detection range of the sensing device was higher than 1.0E-7M 3OC12HSL. At the same time, we also used Hill equation to curve fit the relationship between the time-average GFP expression rate and 3OC12HSL concentration (Fig 2b).
Previous study reported that extracellular concentration of 3OC12HSL is in the range of 1.0E-6 to 1.0E-4M of when P.aeruginosa infection. To verify that the sensing device would be able to sense the natively produced 3OC12HSL by P.aeruginosa, the sensing device was induced by the filtered culture of P.aeruginosa. And the result show that GFP expression rate was 1.159 RFU per OD per minute so that we can estimate the concentration of natively produced 3OC12HSL was 5.0E-7M. This value is higher than 1.0E-7 which confirmed that the sensing device was able to detect the natively produced 3OC12HSL.
Fig1: Schematic of test quorum sensing device (ptetR-LasR-pluxR-GFP). Promoter luxR was activated by LasR-3OC12HSL complex, leading to produce GFP.
Fig2: Characterization results of quorum sensing device. a| GFP production rate per cell over time at different concentration of 3OC12HSL. b| Time-average GFP production rate per cell over time at different concentration of 3OC12HSL.
User Reviews
UNIQ8bbc0b1b1e6c89b5-partinfo-00000002-QINU UNIQ8bbc0b1b1e6c89b5-partinfo-00000003-QINU