Cloning of Chromoprotein Expression Constructs

pUC19 Backbone Preparation

Our experimental workflow commenced with the construction of chromoprotein-expressing E. coli strains. Initially, pUC19 plasmids were transformed into TOP10 E. coli cells via heat shock transformation, followed by selection on LB/ampicillin plates. A single transformant colony was cultured overnight and subjected to miniprep isolation to obtain sufficient pUC19 plasmid DNA.

The purified pUC19 was subsequently linearized through EcoRI restriction digestion. The initial digestion reaction was performed according to the conditions specified below, with a 30-minute incubation period:

However, preliminary transformation results revealed numerous colonies lacking chromoprotein expression, suggesting incomplete vector digestion using the above protocol. To achieve complete linearization, we optimized the digestion conditions by reducing the DNA input and extending the incubation time:

The incubation period was extended to 2 hours to ensure complete digestion.

Insert Preparation

Assembly of chromoprotein inserts into the linearized vector was accomplished using the HiFi DNA Assembly method, facilitated by the generous sponsorship of the HiFi Assembly Kit from New England Biolabs (NEB).

Chromoprotein gene sequences were synthesized as gBlocks by Integrated DNA Technologies (IDT). The following expression constructs were ordered:

• Constitutive mRFP1e expression
• Constitutive aeBlue expression
• Constitutive SYFP2 expression
• Constitutive mRFP1e + aeBlue co-expression
• Constitutive SYFP2 + mRFP1e co-expression
• Constitutive SYFP2 + aeBlue co-expression

Each lyophilized gBlock was resuspended to a final concentration of 100 ng/μL.

HiFi Assembly of Chromoprotein Expression Constructs

Due to the unavailability of spectrophotometric quantification, pUC19 vector concentration was estimated by comparison to a DNA ladder during agarose gel electrophoresis. Assembly reaction ratio was calculated with AI assistance, determining that approximately 5.5-6 μL of insert was required per 2 μL of vector. For consistency, all insert volumes were standardized to 6 μL.

The assembly reaction composition was as follows:

The assembly mixtures were incubated at 55°C for 2 hours to facilitate DNA joining. Following assembly, the reaction products were transformed into chemically competent TOP10 E. coli cells using the standard heat shock transformation protocol. Transformants were selected on LB agar plates supplemented with ampicillin to maintain plasmid selection pressure.

Successful Construction of Chromoprotein-Expressing E. coli Strains

After 24 hours of incubation at 37°C, several pigmented colonies were observed on the transformation plates, confirming the successful construction of chromoprotein-expressing bacterial strains. The appearance of colored colonies provided direct visual evidence of functional chromoprotein expression and proper assembly of the expression constructs.

However, transformants expressing SYFP2 required visualization under blue light excitation to observe the characteristic yellow fluorescence, as reported in previous studies. Since our objective was to identify chromoproteins that produce readily distinguishable colors under ambient lighting conditions for naked-eye detection, SYFP2 did not meet our selection criteria. Consequently, further characterization of SYFP2-expressing strains was discontinued, and efforts were focused on the more visually apparent chromoproteins.

The transformation results are presented in the figures below:

Fig 1. Chromoprotein-expressing bacterial colonies on selective media following transformation. Pigmented colonies displaying distinct coloration patterns are visible on ampicillin-containing agar plates, demonstrating successful expression of the engineered chromoprotein constructs in E. coli TOP10 cells.

Fig 2. SYFP2-expressing E. coli colony visualized under blue light excitation. A single transformant colony exhibits bright yellow fluorescence, confirming successful expression of the SYFP2 fluorescent protein construct.

Comparing the expression of dTomato and mRFP1e

We performed a comparative study of dTomato and mRFP1e expression to determine which chromoprotein provides better visual detection as a reporter protein. Both chromoproteins were expressed using identical genetic regulatory components, including the same promoter, RBS, and terminator sequences. This standardized approach ensured that any differences in expression or coloration could be directly attributed to the specific characteristics of each chromoprotein rather than variations in gene regulation.

Both bacterial cultures were grown under the same conditions at 37°C for 14 hours with shaking at 180 rpm to maintain consistent growth and protein expression environments.

Fig 3. Comparative expression analysis of mRFP1e and dTomato chromoproteins. mRFP1e displays deeper red coloration than dTomato in both agar plate cultures and liquid medium following 14 hours of incubation.

Based on the results shown in Fig. 3, mRFP1e demonstrated better color intensity compared to dTomato under our experimental conditions. The deeper red pigmentation produced by mRFP1e makes it a more suitable candidate for biosensor applications, as stronger visual signals provide better detection sensitivity and easier identification without specialized equipment.

An Interesting Observation from the Coexpression Strain

We noticed something interesting during our experiments that caught our attention. We consistently observed that the mRFP1e+aeBlue coexpression strain produced deeper coloration than the strain expressing mRFP1e alone. This observation sparked our curiosity, so we decided to compare their expression levels directly.

Fig 4. Comparative expression analysis of mRFP1e+aeBlue coexpression and mRFP1e expression strains. The coexpression strain displays deeper red coloration than the mRFP1e-only expression strain in both agar plate cultures and liquid medium following 12 hours of incubation.

The figure above shows cells after 12 hours of expression at 37°C, 180 rpm. We can clearly see that the color intensity of the coexpression strain is noticeably higher than the mRFP1e-only strain in both agar plate cultures and liquid medium. This was quite an interesting finding, though we chose not to pursue its application further. After consulting with our teacher, we learned that expressing two proteins simultaneously may overload the bacterial cells, which could actually decrease the sensitivity of our biosensor system if implemented in the final design.

SDS-PAGE Analysis of mRFP1e and mRFP1e+aeBlue Coexpression

Out of curiosity, we collected cell lysates for further analysis. The gel diagram is shown below.

Fig 5. SDS-PAGE analysis of chromoprotein expression. Cell lysates from mRFP1e and mRFP1e+aeBlue expressing cultures were separated by SDS-PAGE. An additional band of unknown identity is visible in the coexpression sample compared to controls.

The results showed that an unexpected band appears in the lysate of coexpression cells, which is approximately 30 kDa in size and does not match either mRFP1e or aeBlue. We hypothesize that this is due to our use of overlapping start and stop codons at the junction to coexpress the proteins. The stop codon may not function efficiently, leading to poor translation termination and resulting in a larger protein product. Since this was not the focus of our project, we did not conduct any further investigation.

Detecting the best concentration of induction of Tetracycline biosensor

To find out the best concentration of tetracycline (Tc) to induce our biosensor, we performed a simple experiment to see the growth of E. coli in a series of dilutions of Tc.

Concentrations ranging from ~38 ng/mL to 600 ng/mL Tc with 2-fold increments were cultured with E. coli along with a control to observe their growth. After 14 hours of incubation at 37°C, as shown in the figure below, cultures with ~300 ng/mL and ~600 ng/mL Tc showed significant growth inhibition.

Fig 6. Growth inhibition of E. coli at different tetracycline concentrations. E. coli cultures were grown in the presence of tetracycline at concentrations ranging from ~38 ng/mL to 600 ng/mL with 2-fold increments, along with a control (no tetracycline). Significant growth inhibition was observed at ~300 ng/mL and ~600 ng/mL tetracycline concentrations, indicating the cytotoxic effects of higher tetracycline concentrations on bacterial growth.

Based on the above result, we decided to use 200 ng/mL as the maximum detection concentration.

Functional Study on Biosensors

Functional Assays on Salicylate Detection

Design 1 & 2: BBa_25F9SR86 and BBa_25WAI7EY

We designed and ordered two salicylate biosensor constructs, NahR/pSal BBa_25F9SR86 and BBa_25WAI7EY, in pIDT-Amp vector from IDT and pTwist-Amp from Twist Bioscience respectively. The plasmids were then transformed into both TOP10 and BL21 cells for functional testing.

Individual colonies were inoculated into 10 mL LB/Amp starter cultures and grown for 4 hours at 37°C with 180 rpm shaking. We then transferred 100 μL of starter culture into 10 mL fresh LB/Amp medium with salicylate concentrations from 0.1 mM to 2 mM and continued growing at 37°C with 180 rpm shaking. We monitored color changes at 6, 16, 24, and up to 36 hours after induction. At each time point, we collected 1 mL of culture and centrifuged at 8000 g for 1 minute to form cell pellets for better color observation.

Unfortunately, no color changes occurred in any biosensor setup. We tried troubleshooting with different inducer concentrations, longer incubation times, lower temperatures, and different cell strains like BL21. Despite these attempts, we saw no biosensor response, suggesting the problem was with our construct design rather than the experimental conditions.

Functional Assays on Tetracycline Detection

Design 1: BBa_254KMUU6

This biosensor design, similar to the salicylate biosensor above, showed no response to tetracycline (Tc) under any testing conditions, so we moved on to a new design.

Design 2: BBa_252ZMUOK

The tetracycline biosensor successfully detected tetracycline through a visible blue color change due to amilCP reporter gene expression.

In this TetR/pTet biosensor design, after adding 100 ng/mL tetracycline and incubating for 18 hours at 25 °C with 180 rpm shaking, a significant blue color was observed in the biosensor cells. The color became deeper with 200 ng/mL added, indicating a concentration-dependent effect. The controls, including no Tc (0 ng/mL Tc) and pUC19 (added with 100 ng/mL Tc), showed no visible coloration to the naked eye, as shown in the figure below.

However, with 10 ng/mL Tc added, the biosensor didn't show a visible color change indicating no response, meaning the sensitivity of the biosensor is unable to detect this concentration range.

Fig 7. Tetracycline detection by TetR/pTet biosensor (BBa_252ZMUOK). Cell pellets showing concentration-dependent blue color development in response to tetracycline. However, the biosensor shows sensitivity limits with no detectable response at 10 ng/mL concentration.

To further validate the results and better observe the response of the biosensor, we performed another set of experiments using smaller concentration intervals (0, 10, 50, 100, 150, and 200 ng/mL) of Tc to induce the biosensors. The results (Fig. 7) showed that when compared with the empty vector pUC19 control and no Tc control, the cell pellets treated with Tc starting from 50 ng/mL concentration showed a noticeable blue color from amilCP chromoprotein that was visibly different from the controls by naked eye observation. The color intensity increased with increasing Tc concentration.

Fig 8. Another set of replicates using smaller intervals of tetracycline concentrations to assess the response of our biosensor.

However, from this set of results, we observed that the color intensity of the pellets did not resemble that of the first set. This was because we used an overnight culture diluted for the experiment due to time constraints, instead of using a freshly prepared culture. This may have caused an unhealthy status of our biosensors, leading to these results. This made us aware that we should better maintain the biosensor's growth conditions.

Despite these limitations, distinct color variations were still evident between the tetracycline-exposed biosensors and control samples. These findings provide additional confirmation of our biosensor's operational capacity and support the validity of our design approach.

SDS-PAGE validation of the expression

We also performed SDS-PAGE to further validate the expression of the biosensor construct. The whole protein lysate of the biosensor culture was run on a 4-12% Bis-Tris Plus Gel with MES SDS running buffer at 150V. The gel was stained with Coomassie Blue. The protein ladder used was SeeBlue Plus2 Pre-Stained Standard.

Fig 9. SDS-PAGE analysis confirming TetR protein expression in the biosensor construct. A distinct band at ~23 kDa was observed in the biosensor lane, corresponding to the expected molecular weight of TetR protein.

From the gel photo above, when compared with the pUC19 control, a distinct band was observed at ~23 kDa in the lane containing our biosensor, which matches the size of the TetR protein expressed by the biosensor and is absent in the control. This result strongly validates the successful creation of our biosensor.

Bead-Based Biosensor Application

To validate the plausibility of our concept of using alginate beads to immobilize biosensors, we produced beads containing our biosensor and incubated them in LB/Amp culture with or without tetracycline (100 ng/mL or 200 ng/mL). The results are shown below.

Fig 10. Alginate bead-based tetracycline biosensor response. Beads containing biosensor cells or pUC19 control were incubated with varying tetracycline concentrations (0-200 ng/mL) for 16 hours. Visible color changes indicate successful tetracycline detection in the bead format.

The figure demonstrates visually detectable color alterations in beads treated with 100 ng/mL and 200 ng/mL tetracycline-containing medium compared to both pUC19 empty vector controls and tetracycline-free controls following 16-hour incubation. These findings confirm the feasibility of our bead-based biosensor approach.

Functional Study on Degradation Strains

Functional Assays on Salicylate Degradation

For salicylate degradation testing, since we lacked UV-spectrophotometers to measure salicylate or product concentrations, we attempted an alternative approach using FeCl₃·6H₂O, which reacts with salicylate to form purple compounds measurable at 530 nm. However, we could not construct a reliable standard curve, likely due to our expired FeCl₃·6H₂O stock forming Fe³⁺ hydroxide over time. Due to time constraints, we abandoned this quantification approach.

Nevertheless, we ran SDS-PAGE to demonstrate the successful expression of NahG and XylE. The gel diagram is shown below:

Fig 11. SDS-PAGE showing the protein expression in our constructs. In the lane of NahG/XylE expression cells' proteins, two thickened bands can be observed at the positions matching NahG (~49.3 kDa) and XylE (~35.1 kDa).

From the gel results below, we can see that in the lane containing lysates from NahG/XylE-expressing cells, two distinct bands appear at positions corresponding to NahG (~49.3 kDa) and XylE (~35.1 kDa), indicating successful expression of both proteins. Moreover, the XylE band shows noticeably higher intensity than the NahG band, confirming our design concept that XylE is expressed at a higher level than NahG. This likely results in greater XylE activity within the cells, helping to prevent catechol accumulation during salicylate degradation.

Functional Assays on Tetracycline Degradation

A standard curve using absorbance at 360 nm was constructed to measure tetracycline (Tc) concentration in culture medium by spectrophotometer. LB/Amp solution without Tc was used as blank.

Standard LB/Amp solutions with Tc concentrations of 1, 2, 4, 6, 8, and 10 μg/mL were prepared. The absorbance results showed a linear relationship with the equation y = 0.0294x - 0.0041 and R² = 0.999, indicating high reliability. This validates the measurement method for monitoring Tc concentration changes in LB culture medium.

Fig 12. Standard curve for tetracycline concentration measurement. Linear relationship between tetracycline concentration (μg/mL) and absorbance at 360 nm in LB/Amp medium.

The linear equation was then used to calculate Tc concentration in the culture medium for subsequent degradation experiments.

Tetracycline Degradation Assay

Cells expressing TetX were grown in LB/Amp medium for 2 hours. We transferred 100 μL starter culture to 10 mL degradation culture and incubated for 4-6 hours until log phase. Tetracycline was added to final concentrations of ~5 μg/mL and ~10 μg/mL. We immediately measured baseline absorbance (time = 0) by centrifuging cells at 12,000 g for 5 minutes and measuring supernatant absorbance at 360 nm. Subsequent measurements were taken at 2 and 4 hours post-addition.

The results are shown in the tables below, with absorbance values converted into concentration using the standard curve equation.

Fig 13. Time-course analysis of tetracycline degradation by TetX cells over 2 and 4 hours.

The results demonstrated that TetX cells significantly decreased tetracycline concentration in the culture medium, while control setups (pUC19 empty vector cells and LB/amp medium only) maintained their tetracycline levels. This shows that TetX cells are effectively working as expected, with results further supported by SDS-PAGE showing TetX enzyme expression.

We observed that after 4 hours, tetracycline concentration in TetX cultures did not further decrease. We suspect this was due to prolonged incubation, causing cells to enter post-log phase and reduced tetracycline substrate concentration, greatly reducing TetX enzyme activity after 2 hours. To test this hypothesis, we repeated the experiment using a shorter time frame.

Using the same methods as the previous experiment, absorbance changes were measured every 30 minutes over a 2-hour time frame to monitor tetracycline concentration changes in TetX cell cultures compared to controls.

Fig 14. Tetracycline degradation assay over 2 hours. Three conditions were tested: LB only, pUC19 control + tetracycline, and TetX-expressing cells + tetracycline. Error bars represent SD of two biological replicates (n=2).

As shown in the results, the tetracycline concentration in the culture medium of TetX cells continued to decrease at every time point, indicating that TetX is actively degrading tetracycline within this timeframe. This result further confirms the functionality of our tetracycline degradation cells and enhances our understanding of their tetracycline degradation kinetics. In the graph, the error bars represent the standard deviation of two replicates from individual cultures of TetX cells.

In the results above, we observed substantial variation in absorbance values, including initial readings and replicates, despite maintaining careful and consistent technique. We suspect that interference from cells and other components in the LB medium contributed to this variability. Since the reported absorbance 360 nm measuremnt method for tetracycline detection was developed in pure water, it may not fully apply to our conditions. The complex composition of the LB/Amp medium and the presence of cells likely introduced instability in the measurements. Nonetheless, the pronounced decrease in absorbance observed in TetX-expressing strains, compared to the relatively stable control, still provides strong evidence that our degradation cells are functioning effectively.

SDS-PAGE Confirmation of TetX Expression

SDS-PAGE analysis was performed following the protocols outlined in the experiment section. Protein lysates were prepared from the TetX cultures used in the degradation experiments and analyzed alongside lysates from E. coli control and pUC19 empty vector control. The expected molecular weight of TetX protein is approximately 44 kDa.

The SDS-PAGE results clearly demonstrate a distinct band at ~44 kDa in the TetX sample that is not present in either the E. coli or pUC19 control samples. This confirms successful expression of the TetX enzyme in our degradation strain.

Fig 15. SDS-PAGE analysis confirming TetX protein expression in the degradation strain.

Summary of Key Findings

1. We successfully designed and created chromoprotein expression strains for mRFP1e, aeBlue, SYFP2, and a coexpression strain combining mRFP1e and aeBlue, demonstrating their distinct color properties.
2. Our preliminary studies comparing dTomato and mRFP1e chromoproteins revealed that mRFP1e exhibits superior characteristics for biosensor applications due to its faster maturation kinetics.
3. Additionally, when comparing mRFP1e alone versus the mRFP1e and aeBlue coexpression strain, we observed that the coexpression system consistently displayed accelerated maturation and enhanced color intensity, though the underlying mechanism remains unclear.
4. The tetracycline biosensor (BBa_25A81M7M) successfully detected tetracycline presence through visible color changes, with color intensity correlating to tetracycline concentration. This demonstrates the potential for real-world diagnostic applications of these engineered strains.
5. Our tetracycline biosensor cells were effectively immobilized using calcium-alginate encapsulation technology. Viability assays confirmed that the entrapped cells retained their metabolic activity and tetracycline-sensing capabilities, displaying distinct chromoprotein responses upon target detection. This demonstration validates the potential of alginate-based cell immobilization as a practical method for developing portable, contained biosensor platforms suitable for field applications.
6. Our tetracycline degradation remediator (BBa_25CS26NB) strains demonstrated their capability to reduce tetracycline concentrations in the extracellular environment, highlighting their potential as an effective biological approach for mitigating tetracycline pollution in real-world applications.
7. We successfully designed and expressed the NahG/XylE salicylate degradation construct (BBa_257IE4RA). Although we did not directly verify its degradation activity, SDS-PAGE analysis confirmed the expression of both NahG and XylE proteins. Moreover, our results demonstrated that XylE was expressed at a higher level than NahG, supporting our design concept that a higher XylE activity-to-NahG ratio can help prevent the accumulation of the toxic intermediate catechol during salicylate degradation.

These results informed our strategic planning sessions, where we identified key areas for improvement and established a roadmap for project advancement. Our future efforts will prioritize enhancing biosensor sensitivity and resolving the technical limitations we observed. A detailed account of our iterative design process, including the obstacles faced and solutions implemented during biosensor development, can be found on our Engineering page.