Latest revision as of 14:27, 12 October 2023

PobR-Threshold Guard Switch

Sequence and Features

Description

This composite part consists of four main components: the pobR, the pobR RBS, the pobR operator, the pobA/R dual-directional promoter, and the digitizer.

pobR

The gene pobR creates a transcriptional activator that attaches to the pobR operator on the dsDNA before combining with 4-HBA. Once 4-HBA is introduced to the solution, PobR binds with it and triggers the transcription of the dual pobA/R promoter on the side of pobA.

One of the advantages of this protein is its sensitivity and low leakage properties. Even tiny amounts of 4-HBA, at the micromolar level, can trigger transcription. This characteristic is crucial in creating a high-quality digitizer with a sharp response between two stable states^[1].

Additionally, research indicates that most analogs of 4-HBA, such as p-aminobenzoate, can impede the activation of PobR, ensuring precise detection of 4-HBA.

Moreover, the combination of the non-activated PobR and pobR operator will inhibit the transcription of pobR when there is no 4-HBA stimulus, reducing the pressure on our bacteria.

Threshold Guard Switch

The promoters we utilize are controlled by signal-sensitive receptors, which typically demonstrate different relationships between inputs and outputs when exposed to specific inducers.

However, it's important to highlight that a more thorough transcription halt allows for tighter signaling control, which is undoubtedly vital for trace chemical detection. Therefore, We placed the original threshold guard switch (first developed by Ángel & Víctor^[2]) downstream of our detection fragment. Here are the critical components of this post-transcriptional control circuit:

Given PobR's excellent properties, we create a chimeric switch by combining it with a portion of the original threshold guard switch.

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Experiment & Result

1)The Threshold Guard Switch

As described by Ángel and Víctor, the “Digitalizer module” they built has a clearly defined on-and-off status. As mentioned in Design, we used it as a threshold guard switch that only allowed a specific inducer of a specific concentration to open our promoter. To verify the functionality and the minimum threshold of this switch, we conducted a series of gradient concentration tests using classical inducers of the Xyls/Pm system: Benzoic acid and 3MBz.

Inducer: Benzoic acid

Initially, a wide range of 2000µM to 0µM benzoic acid was added to E. coli BL21(DE3) (initial OD value = 0.688) that had the switch sequence (BBa_K3202045). msfGFP fluorescence was measured by a synergy HTX microplate reader with excitation at 485 nm (±20 nm) and emission at 520 nm (±20 nm). Fluorescence was normalized to the OD after the background fluorescence value was subtracted from all RFU data. Continually Measuring every 2 minutes for 12 hours, we surprisingly found that the turning-on threshold of this lay at a low level—between 150 μM and 100 μM.(shown in the figure below)

Besides, the time that this switch needed to turn on was collected when the relative fluorescent units (RFU) reached above 200, providing strong evidence that when the concentration of benzoic acid was above 150μM can be regarded as a line separating the on-and-off status of this switch. However, the response time might not be as promising as we thought. The minimum time our switch cost to reach the on-status was 74.33 minutes at 2000μM Benz. Acid.(shown in the figure below)

Being curious about what happened when benzoic acid concentration changed from 100μM to 150μM, we performed a detailed threshold test around this range (80μM~150μM). Using the same bacteria (initial OD value = 0.480) and following the same protocol, we obtained the detailed situation shown in the figure below. Although the absolute value of RFU might be different from the previous test due to the initial growth status, we still found it distinguishable between the on (above 100 μM) and off (below 100 μM) models of this switch. (shown in the figure below)

Inducer: 3MBz

In order to ensure the on-and-off status that happened on our threshold guard switch is non-specific to benzoic acid, we used 3MBz as another inducer. The result shown below also provided an on-and-off threshold line at 10μM and aligned with the work done by Ángel and Víctor^[2].

Model Result

We simulated T=1000 for each inducer strength, and for each inducer strength we set the repressor strength to be 5-10. It means we simulated $5* 10=10$ times to get 50 points of stable status. We plot a scatter plot to display. And according to the plot we find the threshold to be roughly 0.6 -0.8. As is shown in the following figure.

To unify, we processed the data, took the logarithm of the experimental data to the relative intensity interval, and used the function to fit the data to obtain the fitting curve. Then we find that the true threshold is roughly 680 $\mu mol.L$. Note that we calculate the threshold at which the desired concentration reaches the turn-on threshold, which is $10^0 = 1\mu mol/L$. While at the point where the slope is greatest, that is, $100-150\mu mol/L$, is the threshold our wet lab uses. The repressor strength is more than 10 in terms of the strength interval we divided. Finally, the fitting curve is drawn together with our simulation data through the interval maximum scaling, and it is found that the data fitting is very good.

2)Pobr

Threshold Test

The diagram above shows the changes in GFP fluorescence levels over time for varying concentrations of 4-HBA.

We grew E. coli (Fast-T1) with a chimeric PobR threshold guard switch (GFP version) and measured the quantity of GFP expression over time at different concentrations of 4-HBA using a microplate reader.

After removing the control group and dividing it by OD600, we obtained the following results from our analysis. We observed that concentrations above 0.25mM efficiently activate the switch, leading to a normalized fluorescence intensity more significant than 140 after a certain period. Conversely, intensities below 140 are considered non-activated, as 140 represents the asymptote of the curve with a concentration of 0.25mM, and the proteins produced at that concentration are just enough. Therefore, we conclude that the detection threshold falls between 0.25 and 0.5mM, indicating that it is a trace amount.

We observed negative intensities of normalized fluorescence in some cases. Our analysis indicates that the decrease in bacterial concentration is due to the antibacterial effect of 4-HBA, combined with low GFP production at low concentrations of 4-HBA, resulting in negative intensity.

We believe the detection threshold can be lowered by replacing GFP with *luxI* in the final work. LuxI produces VAI, activating the downstream Quorum sensing system as a signal amplifier.

In addition, We need to analyze how long it takes to open the switch successfully. We define it as open when the intensity level reaches 140.

Time limit

The diagram above shows the time needed to open the switch at different concentrations.

By analyzing the data in the figure, we can observe that a concentration ranging from 0.5mM to 10mM requires approximately 120 minutes. Despite this duration being slightly longer than our initial estimates, it should have minimal impact on the final products because only a tiny quantity of LuxI is required to initiate the quorum sensing system.

References

1. ^[1]Na D, Yoo SM, Chung H, Park H, Park JH, Lee SY (2013) Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nat Biotech 31: 170 – 174
2. ^[2]Calles B , Goni-Moreno A , Lorenzo V D .Digitalizing heterologous gene expression in Gram-negative bacteria with a portable on/off module[J]. 2019.DOI:10.1101/783506.