Results

Preparation of pUC19 Vectors

We began the transformation process by introducing pUC19 plasmids into TOP10 E. coli cells using the heat shock method, followed by selection on ampicillin LB plates. Afterward, plasmid DNA was extracted using a mini-prep procedure, and restriction digestion with EcoRI and PstI was performed to linearize the vector for subsequent HiFi assembly. The linearized plasmids were then purified through gel extraction.

The digestion of the pUC19 vector was performed according to the table below:

Figure 1 shows the gel electrophoresis results matched our expectations. The lanes containing purified, linearized pUC19 plasmid (~2.7 kb) displayed three distinct bands. We selected the plasmids from lane 4 as our candidate for assembly, as we did not have access to a spectrophotometer to measure DNA concentration. We assumed the concentration in lane 3 was at an average level, likely neither too low nor too high, which would avoid negatively impacting the assembly efficiency.

Figure 1: Photograph of result of gel electrophoresis showing the digested and gel purified pUC19 vectors. The first lane is showing the 1kbp+ ladder from NEB.

Cloning of Constructs for Chromoprotein Proteins

Insert preparation

We planned to use the HiFi assembly method to insert our gene constructs into pUC19 vectors, with generous support from NEB through their sponsorship of HiFi assembly kits.

Our gene constructs were synthesized as gBlocks by IDT, and the ordered gBlocks are detailed in the table below:

Next, we diluted the purified fragments to a concentration of 100 ng/µl, following the manufacturer's instructions. We then proceeded with HiFi assembly, utilizing the linearized pUC19 vectors for the process.

HiFi assembly of chromoprotein expression constructs into pUC19

To ensure the success of the HiFi assembly, it is crucial to calculate the molar ratio of the inserts to the vector. According to the manufacturer's protocol, a 2:1 molar ratio (inserts to vector) is recommended for assemblies involving 2-3 fragments, while a 1:1 molar ratio is suggested for 4-6 fragments. However, as previously mentioned, we lack the equipment to accurately measure DNA concentration. Instead, following the advice of our external advisor, we will estimate the concentration by comparing the relative brightness of the DNA bands with that of the ladder bands. This estimation may introduce some uncertainty in the molar ratio calculations.

We set up the assembly following the table below:

Transformation of Recombinant Plasmids

We introduced the assembled plasmids into E. coli using heat shock transformation. The transformed cells were plated on LB/ampicillin agar to assess the functionality of the plasmids in expressing their respective chromoproteins.

Figure 2: E. coli colonies transformed with constitutive chromoprotein expression constructs (cjBlue, tsPurple, amilCP, eforRed, and dTomato) on LB/ampicillin plates.

Figure 3: Cell pellets of E. coli expressing various chromoproteins obtained by centrifugation. Each pellet shows distinct coloration corresponding to the expressed chromoprotein.

In Figure 3, the chromoproteins are ordered from left to right as follows: eforRed, dTomato, amilCP, and tsPurple. cjBlue was not included, as its color development required at least 24 hours, unlike the others, which showed color after 16 hours. Figure 4 shows that cjBlue may not be expressed efficiently, suggesting it may not suit our application.

We suspect the delayed expression of cjBlue may be due to suboptimal conditions or cell health. Since optimizing expression is not our focus, we decided to proceed with the other four chromoproteins.

Figure 4: E. coli cells transformed with the cjBlue expression construct displaying weak chromoprotein production after 16 hours at 37°C.

Optimizing the observation time for Chromoproteins

We investigated the optimal incubation time for chromoproteins, as they require varying durations for maturation and color development. This helps determine if their expression profiles suit our experiments.

We cultured cells expressing dTomato, amilCP, tsPurple, and eforRed in LB/Amp medium at 37°C, shaking at 180 rpm. Samples were taken at 4, 8, 12, 18, and 24 hours, with 1 mL of culture harvested at each point. The cells were pelleted by centrifugation, and the pellets were examined for color development.

At 4 and 8 hours, no significant color was observed. By 12 hours, visible pellets formed with faint coloration. After 18 hours, color intensity increased, suggesting maturation. (See Fig.)

Figure 5: Cell pellets after 12 and 18 hours of incubation at 37°C. The 18-hour pellets display higher color intensity. pUC19 shows cells with the empty vector as a control.

We understand that using a constitutive promoter might not replicate conditions with metal-inducible promoters in later experiments. Nonetheless, these results provide valuable insights into chromoprotein maturation times and expression profiles.

Cloning of Biosensors

For cloning our heavy metal biosensors, we followed the same protocols used for chromoprotein constructs. A constitutive chromoprotein expression setup was included as a positive control, consistently yielding reliable results.

Our biosensor constructs were synthesized by IDT and Twist Bioscience, listed in the table below:

Initial cloning attempts showed few colonies, though the positive control was successful. Colony PCR confirmed the correct insertion of constructs.

Failed Cloning Attempts

Gel electrophoresis validated colony PCR results. Conditions are shown below:

30 cycles of PCR reactions were done in all attempts.

Despite multiple rounds, no distinct bands were observed (Fig. 6), indicating failed cloning. We initiated troubleshooting to identify issues.

Figure 6: Gel result from colony PCR showing absence of bands, indicating no desired insert. Faint bands are primers.

Validation of PCR

We checked the PCR apparatus using validated DNA and primers from a previous project. Tests produced expected results (Fig. 7), confirming PCR apparatus functionality.

Figure 7: Gel image validating PCR conditions. Expected band sizes confirm apparatus functionality, ruling it out as the error source.

Investigation revealed incorrect 5' overlap design in ordered DNA. New orders for corrected sequences were placed with IDT and Twist Bioscience.

Successful Cloning Attempts

With new DNA fragments, we initiated cloning following the previous workflow. The assembly protocol is outlined below. The dTomato construct was used as a positive control.

We successfully obtained colonies for all designs except pCadA and MerR-pMerT. Colony PCR verified correct clones (Fig. 8).

Figure 8:
(a) PbrR construct colony PCR result with expected ~1.5 kbp bands in lanes 3, 4, and 5.
(b) MerR-pCadA construct result with expected ~1.6 kbp bands in lanes 4, 5, and 6.
(c) pZnt and pYodA result with expected ~600 bp bands.

Leaky expression was consistently observed, notably in pZnt and pYodA designs (Fig. 9).

Figure 9: Some clones exhibited leaky expression (visible color without induction). To avoid false positives, we excluded these clones from further study.

Functional Assays on Heavy Metal Detection

We conducted functional assays to test whether our biosensors could respond to the presence of heavy metals. This step was crucial in determining the effectiveness of the constructs and their ability to detect and report the presence of target metals.

The concentrations of heavy metals used in our experiments are listed in the table below:

For all experimental setups, we added 1 mL of the designated metal solution into 19 mL of LB/Ampicillin broth to reach the stated final concentration, with either the corresponding biosensor cells or control cells. This ensured consistent conditions across all experiments, allowing for accurate comparison of the biosensor responses.

I. Lead Biosensor (BBa_K5152004)

The Lead Biosensor Successfully Detects the Presence of Lead Through a Visible Colour Change

In the lead biosensor PbrR-pPbr, upon adding 100 µM of lead and incubating for 12 hours, the experimental setup displayed a clear red coloration in the pellet, indicating dTomato expression. This was in stark contrast to the white pellet observed in the control. Additionally, a faint red hue was visible in the culture medium itself, further confirming successful lead detection by the biosensor (Fig. 10).

Figure 10:
(a): Through naked-eye observation, the cultures expressing the PbrR-pPbr lead biosensor exhibited a distinguishable red coloration, in contrast to the negative control setup where no lead was added.
(b): The red coloration becomes even more prominent in the cell pellet, highlighting the expression of the reporter gene (dTomato). This observation confirms that our biosensor is successfully responding to the presence of lead (Pb).

After 18 hours of incubation, the coloration of the culture medium became distinctly red, clearly indicating a response to lead. However, a faint red hue was also observed in the biosensor cell culture without lead added, suggesting a mild leaky expression effect. This unintended expression in the absence of lead highlights some basal activity of the biosensor system. (Fig. 11)

Figure 11: The PbrR-pPbr lead biosensor shows a significant red coloration after 18 hours of incubation with 100 µM of lead. However, when compared to the pUC19-only control setup, the PbrR-pPbr biosensor without added lead also displayed a mild red coloration, indicating that extended incubation leads to a mild leaky expression of the red chromoprotein.

The Reporter Signal is Dependent on the Concentration of Lead

To investigate whether the reporter signal of the biosensors reflects the concentration of lead, 0.01, 0.1, 1, 10, 500, and 1000 µM lead solutions were added to the biosensor cells. 1 mL of each metal solution was added to 19 mL of LB/Ampicillin broth, containing either the corresponding biosensor cells or control cells, to reach the desired final concentrations.

After 16 hours, the cultures showed no significant differences in colour. However, after 24 hours of incubation, noticeable differences in colour intensity were observed across cultures with varying lead concentrations. In the range of 0.01 µM to 10 µM, the intensity of the red colour increased with higher lead concentrations, indicating a concentration-dependent response.

In contrast, in the 500 µM and 1000 µM setups, the colour became paler, which may be attributed to the toxicity of the high metal concentrations, potentially causing cell death or impairing protein expression. (Fig. 12)

Figure 12: The red colour intensity of the lead biosensor increases with an observable difference as the concentration of lead rises from 0.1 µM to 10 µM. However, when the concentration exceeds 500 µM, there appears to be a negative effect on the colour intensity, potentially due to toxicity or inhibition of cellular functions at high lead concentrations.

From these results, it is evident that our lead biosensor design is highly promising, demonstrating significant potential for further development into a practical and reliable tool for lead detection.

II. Cadmium Biosensor (BBa_K5152006)

We also successfully demonstrated that our MerR-pCadA (BBa_K5152006) cadmium biosensor is capable of detecting the presence of cadmium. Upon adding 200 µM cadmium chloride to the biosensor culture, the treated cells developed a distinct blue coloration, whereas the control cells remained white (Fig. 13). This confirms the functional response of the cadmium biosensor.

Figure 13:
(a): In the pellet form, an obvious blue coloration from the amilCP protein is observed in the pellet of the biosensor culture with 200 µM cadmium added, confirming the activation of the cadmium biosensor.
(b): In the pellet form, an obvious blue coloration from the amilCP protein is observed in the pellet of the biosensor culture with 200 µM cadmium added, confirming the activation of the biosensor.

III. Other Biosensor Designs

For the other biosensor designs, the results did not meet our expectations. The specific issues and functional testing outcomes for these designs are detailed on our Engineering page, which outlines all the processes and challenges we encountered with the remaining biosensor constructs.

Summary of Key Findings:

1. We successfully constructed expression constructs for chromoproteins and validated their expressions. Additionally, we studied the expression time required for the cells in our design.
2. Our PbrR-pPbr (BBa_K5152004) lead biosensor demonstrated promising results, showing significant potential for practical lead detection. Importantly, the reporter signal was found to be concentration-dependent, indicating its sensitivity to varying lead levels.
3. The MerR-pCadA (BBa_K5152006) cadmium biosensor successfully responded to the presence of cadmium, producing a clear colour change, confirming its functionality.
4. All constructs exhibited some degree of leaky expression, emphasizing the need to carefully optimize the timing and conditions for measuring the biosensors' readouts.

Based on these findings, we held discussions to refine our approach and have developed a set of future plans for advancing the project, focusing on improving biosensor performance and addressing the challenges encountered. For further details, please refer to our Engineering page, which provides a comprehensive overview of the processes, challenges, and troubleshooting steps we encountered throughout the development of our biosensor constructs.