Parts Overview

This year, the NOX project has successfully incorporated a fundamental principle of a modular biosensor, resulting in the reconfiguration and reconstruction of several components. These modifications have endowed the biosensor with a high level of compatibility and resilience. A comprehensive analysis was conducted on the Threshold Guard Switch, encompassing thorough characterization and documentation of its constituent elements. Moreover, we documented the orthogonal amplifier kill switch tailored for a semi-contain environment in separate basic parts and composite parts. With the intention of equipping future iGEM teams with valuable information, we are hoping to enable them to effectively incorporate this switch into the construction of resilient and desired circuits in subsequent endeavors.

Our parts collection can be divided into two components, the New Basic Part and the New Composite Parts.

• The new basic parts mainly involve the constituents of detection modules for BPA and bile salts, the mutated LuxR for orthogonal quorum sensing, and the components of our kill switch.
• The new composite parts mainly contain the Threshold Guard Switch, the orthogonal amplifier module, and the kill switch. The Threshold Guard Switch is also selected for our Best Composite Part candidate, which can be directly entered here.

The detailed document of our New Basic Parts and New Composite Parts are listed below.

Basic Parts

Composite Parts

This composite part consists of four main components: the pobR, the pobR RBS, the pobR operator, the pobA/R dual-directional promoter, and the Threshold Guard Switch. The assembly process is as follows: The original pobA/R operon consists of six main components: pobA, pobA RBS, pobA/R dual-directional promoter, pobR operator, pobR RBS, and pobR. The pobA gene encodes an unnecessary enzyme that breaks down 4-HBA. Therefore, we remove the pobA and pobA RBS components but retain the entire promoter, pobR RBS, and pobR.

Best New Composite Part

We are awared the Best New Composite Part prize with our PobR threshold guard switch. The construction of our new composite part combines modeling and experimental verfication and improvement, covering mutiple trial-and-error stages and dimensions.

We have updated the related information here, adding our supplementary experiment apart from the Registry Page.

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 (Na et al., 2013).

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) downstream of our detection fragment. Here are the critical components of this post-transcriptional control circuit:

Schematic Diagram of Threshold Guard Switch (Xyls System)

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

Schematic Diagram Illustrating How We Create the PobR Threshold Guard Switch

For more descriptive information, please visit our Design page for more information.

Experiment Part

The Threshold Guard Switch (Xyls System)

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)

Overall threshold test of guard switch (benzoic acid)

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)

Response time of guard switch (benzoic acid)

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)

Detailed threshold test of guard switch (benzoic acid)

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 (Calles, Goñi-Moreno, & de Lorenzo, 2019).

Overall threshold test of guard switch (3MBz)

PobR Threshold Guard Switch

Determination of the Threshold of Our Switch

We grew E. coli (Fast-T1) with a chimeric PobR Threshold Guard Switch (msfGFP version, BBa_K4619010). We measured the quantity of msfGFP expression over time at different concentrations of 4-HBA using a synergy HTX microplate reader with excitation at 485 nm (±20 nm) and emission at 520 nm (±20 nm). The initial OD600 is 0.9.

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

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 relative fluorescent unit (RFU) 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.

An experiment with smaller concentrations of 4-HBA was conducted to determine the specific threshold of the PobR Threshold Guard Switch. The experiment involved varying concentrations of 4-HBA at 0.6mM, 0.3mM, 0.25mM, and 0.2mM. The results indicate that the expected putative threshold, as predicted in the model, is between 0.2mM and 0.3mM.

Detailed threshold test of the PobR Threshold Guard Switch.

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

Time Needed to Trigger the Switch

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

The diagram shows the time needed to trigger 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.

Specificity of PobR Threshold Guard Switch

We confirmed that our PobR system is specific to 4-HBA by testing its response with other inducer, such as benzoic acid. To demonstrate this, we incubated E. coli with either 5 mM 4-HBA or 5 mM benzoic acid and measured the fluorescence intensity.

Responses of our PobR system to 4-HBA and Benzoic Acid.

The results align with the engineering article cited, demonstrating the superior specificity of our system towards 4-HBA compared to its analogs.

Leakage Control Performance of Our Switch

We conducted an experiment to test the effectiveness of our threshold guard switch in controlling leaks. We incubated two groups of E. coli - one with the threshold guard switch and the other without it. We then measured the A.U. (A.U. = (Intensity of Fluorescence -Basal Fluorescence)/OD600) of msf-GFP emitted by both groups of E. coli at different time when there was no inducer added.

The A.U. of msf-GFP in two groups of E. coli

We observed a significant reduction in leakage with our Threshold Guard Switch. The following photo shows that we can even see the difference between these two groups with our naked eyes.

The fluorescence Photos of these two groups of E. coli.

These results indicate that most of the leakage and false positive phenomena can be eliminated with the help of our threshold guard switch.

Modeling Part

Raw Data Visualization

We drew our experimental data and showed the error bar, and from the bar chart, we could see that there was indeed a threshold.

Bar Graph of PobR

Bar graph of XylS

Data Fit

We perform logarithmic processing on the experimental data, and perform exponential fitting, so that the properties of the switch can be estimated at each concentration, which can be used to predict and establish the switch characteristic curve.

The Switch curve of PobR

The Switch curve of PobR

Bar graph of XylS

T-test and Get the Threshold with the lowest P-value

We conducted the paired T-test on the two adjacent groups respectively and obtained the P value, which was used as a representation of adjacency difference. By detecting the lowest P-value, we determined that our thresholds are located at 0.20-0.21$mM$ and 100$\mu M$, respectively.

The P Value of Adjacement groups of PobR

The P Value of Adjacement groups of XylS

We then draw the threshold correlation curve based on the repressor strength, further indicating the fabulous mathematical characteristics of the switch and its adaptability.

The correlation between the repressor strength and threshold

Reference

1. Na, D., Yoo, S. M., Chung, H., Park, H., Park, J. H., & Lee, S. Y. (2013). Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nature Biotechnology, 31(2), 170-174. doi:10.1038/nbt.2461
2. Calles, B., Goñi-Moreno, Á., & de Lorenzo, V. (2019). Digitalizing heterologous gene expression in Gram-negative bacteria with a portable ON/OFF module. Mol Syst Biol, 15(12), e8777. doi:10.15252/msb.20188777