Engineering Success

During our project experiments, we used engineering principles to organize our work, plan our experiments, and solve problems. We broke the project into smaller, easier-to-handle steps, which let us repeat the engineering process multiple times and improve our methods along the way.

This organized approach was really helpful when our experiments didn't work and we needed to figure out what went wrong. In the next section, we'll show how we successfully completed our project by using engineering design principles and continuously going through cycles of designing, building, testing, learning, and redesigning.

Cycle I: Design

Identifying the local problem and solution

At the beginning of our project, our team met to brainstorm local problems that could be solved using synthetic biology. We identified pharmaceutical waste in Hong Kong's water sources as a significant issue. Through background research and interviews with various groups - including industry representatives, citizens, farmers, aquaculture farmers, medical professionals, environmental professionals, and government officials - we confirmed that pharmaceutical wastewater pollution is a real problem in Hong Kong.

While Hong Kong has comprehensive regulations and systems for pharmaceutical waste disposal plus effective wastewater treatment, there are known cases of misuse and regulatory violations by different parties, including ordinary citizens, often due to a lack of awareness or education. This background research emphasized the importance of developing solutions for this problem and using synthetic biology to educate the public about the issue.

Through further research, we discovered that tetracycline, an antibiotic commonly used in aquaculture and pig farming, and salicylate, a derivative of aspirin (a common painkiller and anti-inflammatory drug), are frequently found contaminants in Hong Kong's water bodies, including the Kai Tak River, where residues of these compounds have been reported. Although tetracycline is found to be in very low concentrations in the ocean and main rivers of Hong Kong and China, it has potential for bioaccumulation. Furthermore, as our research found out, there are people such as aquaculture farmers using tetracycline as antibiotics to treat fish diseases while not following the guidelines and causing point pollution, which creates a problem.

Consequently, our team's project aims to develop methods to both detect and degrade these two contaminants using the power of synthetic biology.

(Chui et al., 2020; Chung et al., 2018; Minh et al., 2009; Wilkinson et al., 2022; Leung et al., 2013; Economist Impact, 2024)

Project Structure

After confirming our project goals, we divided our project into four main components: Salicylate Biosensor Tetracycline Biosensor Salicylate Degradation Tetracycline Degradation. We started our experimental work by focusing on designing the two biosensors, which helped us get familiar with lab techniques and routines since we were high school students experiencing a real research project and using advanced synthetic biology techniques for the first time.

Chromoprotein Selection and Characterization for Biosensor Development

The first focus of our project was developing biosensors with an emphasis on accessibility, making chromoproteins with visible readouts that don't require special equipment or techniques, particularly valuable for our applications. To optimize our reporter gene selection, we began our experimental plan with a comprehensive study of chromoprotein candidates.

While our previous teammates successfully used dTomato and amilCP as reporter genes, we questioned whether these were the best choices for our current goals. Therefore, this year we expanded our characterization to include three additional candidates: aeBlue (BBa_K864401), mRFP1e (engineered version of BBa_E1010), and SYFP2 (BBa_K864100).

We designed expression constructs for each chromoprotein, with particular interest in mRFP1e and aeBlue due to their red and blue colors, respectively. These high-contrast colors improve visual detection with the naked eye, making them ideal for accessible biosensor applications. Although our previous dTomato and amilCP selections also provided red and blue outputs, literature reports indicate that mRFP1e significantly reduces expression burden on host cells. (Bao et al., 2020) This characteristic theoretically promotes both improved growth rates and increased protein expression levels, potentially enhancing biosensor sensitivity.

Additionally, amilCP presents practical limitations in experiments, requiring low temperatures (below 30°C) and extended maturation periods (greater than 16 hours) to achieve full color development. In contrast, aeBlue demonstrates more favorable characteristics, requiring less restrictive environmental conditions and achieving color maturation in shorter timeframes according to published reports.

This chromoprotein characterization study served a another purpose in our project development. Since our team recently learned fundamental biotechnology techniques, including pipetting and cell culturing, working with chromoprotein expression constructs provided an excellent training platform. The obvious visual readouts from color changes in transformed cells allowed us to easily assess whether our experimental techniques and designs were working correctly. This approach enabled us to distinguish between technical execution issues and gene design problems before proceeding to our more complex biosensor constructs, where troubleshooting would be significantly more challenging.

Chromoprotein Expression Construct Design and Co-expression Strategy

We designed expression constructs for mRFP1e, aeBlue, and SYFP2 using standardized iGEM BioBrick parts. Our constructs incorporated a strong constitutive promoter (BBa_J23100), a strong ribosome binding site (BBa_B0034), and a double terminator (BBa_B0015) to ensure strong and constitutive protein expression.

During the design process, we became curious about an intriguing question: what visual outcome would result if host cells co-expressed two different colored chromoproteins? After obtaining permission from our teacher, we decided to investigate this phenomenon experimentally. Consequently, we designed three additional co-expression constructs containing the following combinations: mRFP1e + aeBlue, mRFP1e + SYFP2, and aeBlue + SYFP2.

This co-expression approach introduced us to an important concept in synthetic biology gene design. Initially, we assumed that co-expressing two proteins would require complete, separate expression units for each gene, including individual promoters, ribosome binding sites, and terminators. However, further research revealed a more elegant and effective strategy commonly employed in synthetic biology applications.

We learned that optimal co-expression can be achieved by directly linking the stop codon of the first coding sequence with the start codon of the second sequence, creating a TAATG junction on the coding strand. This design strategy enables simultaneous translation of both proteins from a single transcript, providing better control over the relative expression levels of the two chromoproteins compared to using separate expression cassettes.

By implementing this approach in our three co-expression constructs, we aimed to investigate the resulting color phenotypes while gaining valuable experience with polycistronic expression design principles that are widely used in synthetic biology applications.

Cycle I: Build

Cloning Strategy and Chromoprotein Expression Analysis

We ordered our chromoprotein constructs as gene fragments rather than pre-assembled plasmids. Although direct plasmid ordering would have been more convenient, we chose this approach to practice essential laboratory skills and gain experience with experimental design, as mentioned previously. The gene fragments were synthesized by IDT and cloned into pUC19 vectors at the EcoRI restriction site using the HiFi assembly method (we thank NEB for sponsoring iGEM teams with these kits).

Initial Cloning Challenges and Troubleshooting

Our first cloning attempt was unsuccessful. Following heat shock transformation and ampicillin selection, numerous colonies appeared on the plates, but none displayed the expected chromoprotein coloration. To diagnose this issue, we performed colony PCR using M13 primers to determine whether any colonies contained our genes of interest. The absence of PCR bands indicated that the colonies were likely false positives, resulting from incomplete EcoRI digestion of the pUC19 vector, as suggested by our teacher. Consequently, host cells containing undigested pUC19 retained ampicillin resistance and formed colonies without harboring our constructs.

For our second attempt, we modified the protocol by extending the pUC19 linearization digestion time from 30 minutes to 2 hours and reducing the amount of vector DNA used (sample volume used from 20 µL (from the miniprep) to 10 µL). Since we lacked access to a nanodrop or spectrophotometer, precise DNA concentration measurements were not available. After ~24 hours of incubation, we successfully observed colored colonies, confirming successful chromoprotein expression strain creation.

Cycle I: Test

SYFP2 Expression Characteristics

Notably, SYFP2 constructs and co-expression strains containing SYFP2 showed no visible coloration, despite literature reports describing yellow-colored SYFP2 colonies. However, examination under blue light revealed bright fluorescence in some colonies, indicating successful SYFP2 expression. The protein exhibited predominantly fluorescent rather than chromogenic properties under our experimental conditions. The co-expression constructs containing SYFP2 showed neither coloration nor fluorescence, but since this was not our primary focus, we did not pursue further investigation into the reason for the failure of expression.

Comparative Expression Analysis: dTomato vs. mRFP1e

We proceeded to compare dTomato and mRFP1e expression to evaluate whether mRFP1e truly reduces cellular expression burden and enhances growth as reported in the literature. Both constructs utilized identical pUC19 plasmid vectors with the same promoters, ribosome binding sites, and terminators, and were transformed into TOP10 E. coli strains under identical conditions.

From the results below, no matter on agar plate or culture medium, the mRFP1e expression cells showed a much deeper red coloration when compared with dTomato after 14 hours of incubation under 37°C (180 rpm for culture). This shows that mRFP1e is a more suitable candidate as a reporter gene.

Unexpected Co-expression Phenotype

An interesting observation emerged from the mRFP1e and aeBlue co-expression experiments. Colonies consistently displayed darker, deeper red coloration compared to mRFP1e-only constructs. Contrary to our expectation of observing purple coloration (red plus blue), the colonies showed no resemblance to aeBlue's characteristic color. This enhanced red phenotype appeared consistently across both agar plates and liquid cultures, with co-expression strains developing deeper, faster coloration within 12 hours compared to single-protein constructs. However, after >20 hours of incubation, the color of mRFP1e and the co-expression construct becomes the same.

We hypothesized that co-expressing two proteins might intensify the apparent coloration, leading us to consider this approach for our biosensor reporter system. However, after consulting with our teacher, we learned that co-expression increases cellular metabolic burden. In biosensor applications, this additional workload could potentially overload cells and interfere with their ability to detect target substances. Consequently, we abandoned this approach and did not pursue further investigation into the mechanism underlying enhanced coloration in co-expression systems.

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.

The gel above showed that in the lane containing mRFP1e+aeBlue coexpression cell lysates, an unknown band appeared when compared to the controls, and the band size does not resemble any of the chromoproteins being expressed. Since this was not the focus of our project, we did not investigate it further. We can only speculate that it may be due to incomplete translation termination in the first coding sequence of our coexpression construct, possibly caused by the overlapping start and stop codons at the junction between the first and second coding sequences, resulting in a slightly larger mRFP1e protein product.

Cycle I: Learn

From the above cycle, we learned several important conclusions:

Transition to Biosensor module and Degradation module Development

Having gained familiarity with these fundamental techniques, we proceeded to the major component of our project: designing biosensors and degradation modules. According to our original plan, we intended to separately test the biosensor inducible module and degradation modules before combining them into an integrated biosensor system with inducible degradation capabilities. This marks the start of our next engineering cycle:

Cycle II: Design

First Biosensor Module Design

First Tetracycline Biosensor Design (BBa_254KMUU6)

For the tetracycline detection system, we utilized the well-characterized TetR/pTet regulatory system from iGEM parts BBa_R0040 and BBa_C0040. This system serves as a reliable foundation for inducible expression applications. Our construct design incorporated a strong constitutive promoter (BBa_J23100) to drive TetR expression, paired with medium-strength RBS (BBa_B0032). The TetR coding sequence (BBa_25ZXMHWA) was codon optimized for E. coli expression.

We used the medium-strength RBS strategically to balance TetR expression levels. Since TetR functions as a repressor protein, excessive expression could reduce biosensor sensitivity by over-repressing the reporter system. Conversely, insufficient TetR expression would result in high basal expression levels, causing false-positive results that compromise detection accuracy.

The construct included the pTet promoter sequence from BBa_R0040, followed by mRFP1e as the reporter gene. We chose mRFP1e based on our previous experimental results demonstrating superior and faster color development under our laboratory conditions. The construct was completed with a double terminator (BBa_B0015) to ensure proper transcriptional termination.

First Salicylate Biosensor Design and Challenges (BBa_25F9SR86)

For salicylate detection, we employed the NahR/pSal regulatory system using iGEM part BBa_J61051. Although this part has been frequently used and characterized within the iGEM community, we encountered a significant documentation issue: the exact locations and boundaries of individual components (NahR coding sequence, pSal promoter, and NahR expression elements) within BBa_J61051 were not clearly defined.

To address this limitation, we attempted to identify and redesign individual components to potentially enhance salicylate-induced sensitivity. In the original BBa_J61051, the entire NahR expression unit was oriented in the antisense direction. Our first design approach involved reverse complementing this sequence to the sense orientation, facilitating easier handling and reducing potential confusion during construct assembly.

We identified the NahR reading frame using Benchling's translation tool to locate start and stop codons. For NahR expression, we incorporated a strong constitutive promoter (BBa_J23100), medium-strength RBS (BBa_B0032), and a double terminator ((BBa_B0015)). Our strategy aimed to moderate NahR expression levels, preventing excessive activation that could increase basal pSal promoter activity and reduce biosensor specificity.

Critical Design Decisions and Subsequent Issues

When identifying the pSal promoter and NahR binding site regions, we discovered that different iGEM teams had used varying sequence lengths from BBa_J61051, creating uncertainty about the optimal boundaries. Unfortunately, we made a decision we later regretted: prioritizing construct size minimization, we excluded portions of the sequence that may have been essential for proper function, potentially explaining our first design's failure.

After extracting what we believed to be the NahR binding site and pSal promoter region, we added strong RBS (BBa_B0034), amilCP (BBa_K592009) as the chromoprotein reporter gene, and double terminator (BBa_B0015). We subsequently realized a significant design flaw: using identical terminators for both genes within a single construct. This decision likely contributed to the obstacles and delays we encountered during gene synthesis, as identical sequences can cause synthesis and assembly complications.

Degradation Module Design

Tetracycline Degradation Construct (BBa_25CS26NB)

Through literature review, we identified TetX, a flavin-dependent monooxygenase, as an effective enzyme for tetracycline degradation. TetX breaks down tetracycline into 2-acetyl-2-decarboxamido-tetracycline, a non-toxic, non-antibiotic final product. This single-enzyme solution significantly simplified our experimental design compared to multi-step degradation pathways.

Literature reports confirmed that TetX can be successfully expressed, properly folded, and functionally active in E. coli cells, enhancing bacterial tetracycline resistance. Additionally, since tetracycline naturally enters E. coli cells (which contributes to its antibiotic mechanism), the TetX enzyme does not require secretion to effectively degrade extracellular tetracycline. (Yang, Moore, Koteva, Bareich, Hughes, & Wright, 2004)

Our construct design incorporated a strong constitutive promoter (BBa_J23100) and strong RBS (BBa_B0034) to maximize enzyme expression levels and enhance degradation efficiency. We also codon optimized the TetX coding sequence (BBa_25ZXMHWA) for E. coli expression. We later discovered that pET protein expression systems would have provided even stronger expression in E. coli, but this insight came too late in our design process to implement.

Salicylate Degradation Construct Design (BBa_257IE4RA)

Salicylate degradation required a more complex approach due to the toxicity of intermediate products. NahG enzyme converts salicylate into catechol, and since salicylate can enter E. coli cells naturally, no secretory tags were necessary. However, catechol itself is ecotoxic, meaning that simply degrading salicylate would solve one environmental problem while creating another.

To address this issue, we incorporated XylE enzyme, which converts catechol into non-toxic metabolites according to literature reports. This two-enzyme system required co-expression strategies, building upon our experience with chromoprotein co-expression experiments.

We designed two different co-expression approaches.

The first design used a strong constitutive promoter (BBa_J23100) and two RBSs to drive the expression of both NahG and XylE coding sequences. Since we know that the intermediate product catechol is toxic waste, we don't want it to accumulate in the system, so we designed the expression of XylE to be stronger than that of NahG, ensuring that the breakdown rate of catechol by XylE would exceed its formation by NahG. To achieve this, we used a medium-strength RBS (BBa_B0032) for NahG and a strong RBS (BBa_B0034) for XylE, with a double terminator (BBa_B0015) added at the end.

For the second approach, we conducted additional research and discovered the possibility of creating fusion proteins using linker sequences. This strategy would produce a single large protein containing both enzyme subunits within one polypeptide chain. Intrigued by this concept and curious about its feasibility for our application, we designed a construct to test this fusion protein approach alongside our traditional co-expression method. However, this design could not be synthesized by any company that we approached, so we could not make any progress with this design.

Cycle II: Build

Plasmid Synthesis Difficulties

We initially ordered our constructs as plasmids from IDT in pIDT-Amp vectors. Our bulk order included all the biosensors and degradation constructs. However, all of the degradation constructs failed to be synthesized, forcing us to immediately modify our design sequences, optimize them, and place a new order with Twist Bioscience. In the meantime, we could only focus on testing our biosensor constructs.

Cycle II: Test

Establishing a Concentration for Tet Functional Study

Since tetracycline is an antibiotic that inhibits and kills E. coli, our host, we need to know which concentration is suitable for the induction of our biosensor while not killing our cells. Actually, we thought about using a tetracycline-resistant strain, but we found out that the resistance mechanism in E. coli was actually due to an active pump which pumps out tetracycline from inside the cells, so this approach would definitely interfere with our biosensor which relies on tetracycline inside our cells to bind to the TetR protein.

Therefore, we carried out a growth study with different concentrations of tetracycline. At that time, since we did not have access to a spectrophotometer, we could not measure the OD, but we judged the growth of the cells by the cloudiness of the culture. We carried out a serial dilution of tetracycline with a concentration range of ~600 ng/ml to ~38 ng/ml. Although the MIC of E. coli was reported to be 10 μg/ml, we found that at ~300 ng/ml, the growth of the cells was significantly affected after 14 hours, as shown in the diagram below, where the growth only shows after 22 hours, so it is not suitable for our experiment. Therefore, we decided that 200 ng/ml would be the highest concentration for induction.

First Functional Testing Trial on Biosensors

After receiving our first batch of plasmids, we transformed plasmids with biosensor constructs - Tetracycline Biosensor (BBa_254KMUU6) and Salicylate Biosensor (BBa_25F9SR86) into E. coli TOP10 strains and performed ampicillin selection. We obtained colonies and proceeded with functional testing using the following methods:

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 containing the respective inducers. For tetracycline testing, we tried concentrations within the normal induction range of 1 ng/mL to 200 ng/mL. For salicylate, we tried concentrations from 0.1 mM to 2 mM.

We monitored color changes at 6, 16, 24, and up to 36 hours post-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, comparing induced cultures to uninduced controls and normal cells with inducer.

Unfortunately, no observable changes occurred in any biosensor setup. We attempted troubleshooting by testing various conditions, including different inducer concentration ranges, extended incubation times, reduced incubation temperatures, and alternative cell strains such as BL21. Despite these modifications, we observed no biosensor response, leading us to conclude that the issue was more likely originated from our construct design, necessitating design revisions.

Cycle II: Learn

This concludes this engineering cycle that focused on the design, build, and testing of our biosensor designs, and we gained experience that:

To conclude from our experimental results of functional studies on our biosensors:

Therefore, in the next cycle, we will redesign and test the biosensor constructs using the experience we learned in this cycle. Meanwhile, we brainstormed and consulted different experts, such as our teachers, and we hypothesized some reasons for the failure in our biosensor designs, which we will incorporate as corrections in our next cycle.

Cycle III: Design

New Tetracycline Biosensor Design Modifications and Rationale (BBa_252ZMUOK)

For our new tetracycline biosensor design, we suspected that the previous construct's failure was due to the strong promoter that we used to drive TetR expression, which is a repressor of pTet transcription. Especially when we were using the vector pIDT-AMP, which is a high-copy plasmid, it causes a very high level of transcription. Therefore, it may cause the TetR repression effect to be too strong. Meanwhile, in the tetracycline functional study, we cannot increase the tetracycline concentration due to its inhibitory effect on E. coli growth, so the induction concentration must be kept low. Therefore, in the next design, we changed the promoter to a medium-strength constitutive promoter (BBa_J23111), hoping this will solve the problem.

Also, we changed the reporter gene from mRFP1e to amilCP to align with our salicylate biosensor design. This modification was particularly advantageous because amilCP folds more efficiently at lower culturing temperatures (below 30 °C). This enables future experiments where both biosensor types can be cultured together in the same incubator. Moreover, it reflects real-world conditions, as environmental temperatures—especially in aquatic habitats—are typically below 30 °C.

New Salicylate Biosensor Design Modifications and Rationale (BBa_25WAI7EY)

For our revised salicylate biosensor design, we hypothesized that the previous failure stemmed from altering the original sequence orientation. In the original BioBrick part, the NahR expression unit was positioned in an antisense orientation, but our initial design modified this to the sense direction. This change may have caused transcriptional interference between the NahR expression machinery and the reporter gene unit, ultimately leading to our first design's failure. To address this issue, we reverted the NahR expression module back to its original antisense orientation while keeping all other design elements unchanged.

Cycle III: Build

Second Synthesis Attempt and Extended Delays

For our second trial, we ordered redesigned biosensor constructs and degradation modules from Twist Bioscience as plasmids in pTwist-Amp vectors. Unfortunately, the delivery time far exceeded our expectations. Despite ordering in July, the constructs only arrived in late September. Moreover, synthesis success was limited: only the redesigned NahR/pSal salicylate biosensor and TetX tetracycline degradation constructs could be successfully synthesized, while all others failed.

Lessons Learned and Budget Constraints

This experience taught us that while ordering plasmids directly initially seemed more efficient and cost-effective, we had underestimated the difficulties and time costs associated with plasmid synthesis. Significant project time was lost waiting for deliveries or discovering synthesis failures after extended waiting periods.

Upon learning that several constructs could not be synthesized, we immediately placed orders with BGI company. However, budget limitations forced us to order plasmids individually rather than in bulk, causing additional delays. The same synthesis problems recurred, with many plasmids failing to be successfully cloned.

This experience highlighted the importance of better planning and preparation for such contingencies in future projects. We finally received functional constructs in October, significantly impacting our project timeline.

Initial Construct Validation

For the biosensor modules, the redesigned construct was ordered from Twist Bioscience in pTwist-AMP vector and transformed into both TOP10 and BL21 E. coli host strains. Following ampicillin selection, we obtained four colonies and proceeded to functional studies.

For the degradation modules, after ordering from three different companies, we finally obtained the TetX and NahG/XylE degradation constructs. However, the fusion protein design could not be synthesized by any company. Additionally, due to technical difficulties, the NahG/XylE construct could only be synthesized in an intermediate plasmid (pKMV) containing a kanamycin resistance gene, requiring all downstream processing to be performed with kanamycin selection rather than ampicillin.

Both degradation constructs were transformed into E. coli TOP10 and BL21 cells and selected on ampicillin or kanamycin LB agar plates respectively.

Cycle III: Test

Second Biosensor Trial Results

Tetracycline Biosensor Functional Assay

This time, our redesigned construct (BBa_252ZMUOK) performed as expected. Cultures treated with 100 ng/mL tetracycline (final concentration) began showing blue coloration from amilCP reporter protein expression. When the tetracycline concentration was increased to 200 ng/mL, color intensity correspondingly increased, while control cells showed no significant blue coloration. The blue coloration was clearly visible in the culture medium and became even more pronounced after centrifugation when cells were pelleted.

However, at 10 ng/mL tetracycline, no significant color difference was observed compared to the uninduced control, indicating a detection threshold between 10 and 100 ng/mL under our experimental conditions. The results are shown in the diagram below.

Learning from the above results, we decided to use smaller concentration intervals to better observe the response of our biosensor. The results are shown below.

From these results, we can see that the color intensity of this set does not resemble the first set, even though the growth conditions of the two experiments were the same. The difference was that we used overnight culture of the biosensor and diluted it in fresh LB/Amp directly instead of preparing a new starter culture, as we did not have sufficient time for the experiment (wiki freeze was approaching in two days). However, this result taught us that optimizing cell conditions is critical for biosensor performance, and we should carefully control the growth conditions of the cells.

Another noteworthy observation is that we found a mild leaky expression problem, since the 0 ng/mL biosensor pellet shows a noticeable color difference compared to the pUC19 empty vector control. This indicates that we need to better modify and adjust the gene regulation and biosensor conditions.

Nevertheless, we can still observe differences in color intensity between tetracycline-treated biosensors and the controls. This set of results further validates the functionality of our biosensor and proves our design concept.

SDS-PAGE validation of the expression

We also performed SDS-PAGE analysis to validate protein expression in our constructs. As shown in the gel image below, the lane containing TetR/pTet expressing cells displays a prominent band at approximately 23.3 kDa. However, we must consider that amilCP has a molecular weight of ~25.6 kDa (visible in the amilCP expressing cell lane), which means any leaky expression of amilCP could potentially overlap and interfere with our results.

Despite this potential complication, our functional studies successfully demonstrated that the construct can detect tetracycline. Additionally, when comparing the gel bands, the TetR/pTet lane shows a thicker band positioned lower than the amilCP control, indicating a smaller protein size relative to amilCP. Based on these observations, we conclude that the observed band primarily represents TetR expression rather than amilCP leakage.

Bead-Based Biosensor Application

As we proposed, using alginate beads to immobilize the biosensor would be an effective way to utilize the biosensors, since this approach reduces the risk of culture spillage, improves viability, enables easier observation, and better maintains the environment for the biosensor cells. (Massana Roquero & Katz, 2022) After confirming the functionality of the biosensors, we tested them in alginate beads. The results are shown below.

As shown in the figure, a color change distinguishable by the naked eye can be observed in beads exposed to 100 ng/mL and 200 ng/mL tetracycline culture medium when compared with pUC19 empty vector control and no Tc added control after 16 hours. This again provides direct evidence that our concept of using beads for biosensor applications is viable.

Salicylate Biosensor Functional Assay

We proceeded with functional testing on the new design (BBa_25WAI7EY) using the following protocol: selected colonies were incubated in LB/Amp starter culture for 2 hours, then transferred to fresh LB/Amp medium at 1:100 dilution and incubated for an additional 4 hours. We added 100 mM sodium salicylate solution to achieve a final concentration of 1 mM in the culture medium.

Despite multiple attempts and monitoring at 6, 12, and 18 hours post-induction, we observed no significant color changes in our biosensor cultures. This result indicated that our redesigned construct, while showing improved basal reporter expression, still lacked functional salicylate responsiveness. The persistent lack of an inducible response suggested that additional design modifications would be necessary to achieve a working salicylate biosensor.

We then immediately designed a third version, suspecting that the earlier failures were due to truncating part of the pSal promoter or the NahR regulatory sites from the original BBa_J61051 part. Therefore, in the third design, we included the complete sequence of BBa_J61051 from just before the start codon of the NahR coding sequence until the last base of the part. This may include the intrinsic Pr promoter sequence and RBS, so we added the BBa_J23100 promoter and BBa_B0034 RBS upstream of these elements to drive stronger NahR expression. However, at the time of writing this report (with only a few days remaining before the wiki freeze), the plasmid had not yet arrived, forcing us to abandon testing of this design.

First Trial of Degradation Constructs

Tetracycline Degradation Assay

For tetracycline degradation testing, as we learned that TetX can also improve survivability, we decided to try with a higher concentration of tetracycline up to 10 μg/ml (The MIC of E. coli), as also suggested by our AI helper. Since this can help us to easily distinguish if our cells have successfully expressed TetX from control cells, the growth difference can be significant. Thus, a 5 μg/ml and 10 μg/ml concentration of tetracycline were used to see the performance of our tetracycline degradation cells.

We found that tetracycline could be detected at an absorbance of 360 nm using a standard spectrophotometer, which allowed us to generate a standard curve with tetracycline solutions prepared in LB medium. (Ivantsova et al., 2025) Since our lab does not have a spectrophotometer, we are grateful to our chemistry teachers for lending us their lab’s instrument for this experiment.

Tetracycline standard curve ABS360

We plotted a standard curve of absorbance at 360 nm against standard tetracycline solutions in the range of 1 μg/ml to 10 μg/ml. LB/Amp solution was used as the solvent and blank solution. The standard curve and the linear equation are shown below. The R² of the curve is 0.999 which shows that it's highly reliable.This standard curve proved that this method of measurement is valid and it can reflect the concentration of tetracycline in the culture medium.

Degradation Protocol and Results

Transformant colonies were selected from ampicillin plates and grown in LB/Amp medium for 2 hours. We then transferred 100 μL starter culture to 10 mL degradation culture and incubated for 4-6 hours until log phase. Tetracycline was added to a final concentration of ~5 μg/mL and ~10 μg/mL, and 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.

Results showed that cells expressing TetX significantly reduced absorbance at 360 nm compared to normal E. coli containing pUC19 after 2 hours, indicating decreased tetracycline concentrations in the medium. Additionally, TetX-expressing cells showed larger cell pellet sizes and higher OD600 values compared to pUC19 controls, further supporting our hypothesis of enhanced survival and growth, as shown in the diagram below. However, at the 4 hour time point, the value has not shown further change when compared to the 2 hour time point. This makes us wonder why. We hypothesized that it is due to the decrease of tetracycline to a very low concentration which hinders the enzymatic reaction rate making it slow, and also the E. coli cells may have reached a post-log phase, where they are not in a healthy status, so their energy and condition are no longer optimal for the enzymes to work.

Given these results, we subsequently conducted measurements within a 2-hour timeframe to optimize monitoring of tetracycline degradation by our cells. Measurements were taken at 30-minute intervals to better track the degradation activity during the period of highest enzymatic activity.

The results graph shown above demonstrates that the tetracycline concentration decreases continuously at 30-minute intervals throughout the measured timeframe. This data further confirms the functionality of our engineered cells and enhances our understanding of their tetracycline degradation kinetics.

In our results, we observed considerable variation in absorbance values—including the initial readings and replicates—despite careful attention to technique. We suspect this variability arises from interference by cells and other components in the LB medium. The reported ABS360 method for tetracycline quantification was developed using pure water, which differs from our experimental conditions. The complex composition of the LB/Amp medium and the presence of cells likely make this measurement less stable. Nevertheless, the clear decrease in absorbance for TetX-expressing strains, compared to the relatively stable control, strongly supports the effectiveness of our degradation cells.

Protein Expression Validation

The TetX expression was also confirmed by SDS-PAGE. The expected ~44 kDa TetX band matched the size.

Salicylate Degradation Assay Challenges

Experimental Limitations

For salicylate degradation testing, we planned to add salicylate to a final concentration of 0.5 mM to cultures containing the NahG/XylE co-expression construct to study the degradation effect. However, we faced significant limitations due to lack of sophisticated equipment such as UV-spectrophotometers with 230nm wavelength needed to measure concentrations of salicylate, catechol, or the final product (2-hydroxymuconic semialdehyde).

Alternative Detection Method

Research revealed that FeCl₃·6H₂O reacts with salicylate to form purple compounds quantifiable by measuring absorbance at 530 nm using our conventional spectrophotometer. We attempted to construct a standard curve using FeCl₃·6H₂O and standard salicylate solutions in LB medium.

Technical Difficulties

Unfortunately, we were unable to construct a reliable standard curve even we tried several times. We suspected that our FeCl₃·6H₂O stock had expired, as literature indicated that this chemical forms Fe³⁺ hydroxide over time, preventing accurate standard solution preparation. Due to time constraints, we were forced to abandon this approach for salicylate degradation quantification.

Protein Expression Validation

Nevertheless, we also studied if the NahG and XylE proteins were successfully expressed by our construct design. In the protein gel below, NahG (~49.3 kDa) and XylE (~35.1 kDa) matching size bands were observed in the gel photo, which indicates the successful expression.

Importantly, the band corresponding to XylE shows noticeably higher intensity than that of NahG. This confirms our design concept: XylE is expressed at a higher level than NahG, which could result in greater XylE activity within cells carrying this construct and help prevent catechol accumulation during salicylate degradation.

Cycle III: Learn

Summary

This concludes our project. Through the experimental engineering cycles, we gained valuable insights into our work. We learned that employing an effective and systematic problem-solving approach was essential to successfully cloning our constructs and conducting investigations on our designs. Each iteration of the cycle allowed us to refine our methods, troubleshoot issues, and adapt our experiments based on the challenges we encountered, ultimately leading to a deeper understanding of both the engineering process and the functionality of our biosensors. Particularly due to our inexperience and inadequate planning, we were fortunate that we could obtain experimental results just a few days before the wiki freeze. This experience emphasizes the importance of better planning and preparation.

From our experimental results, we can conclude the following for our project:

With these results and conclusions, we have successfully demonstrated that our concept is viable and proven that our tetracycline biosensor and degradation designs can function as intended. However, we are fully aware that significant room for improvement exists and that further investigations are necessary to refine the system.

Limitations and Areas for Improvement

Several major considerations need to be addressed in our project:

Therefore, we are beginning a new round of the engineering cycle to address these important points. By combining our biological system with the device, we aim to improve our product's functionality, applicability, and safety. It is crucial for us to thoroughly investigate every aspect to ensure that our system meets the necessary standards and regulations for real-world implementation.

Major References

Bao, L., Menon, P. N. K., Liljeruhm, J., & Forster, A. C. (2020). Overcoming chromoprotein limitations by engineering a red fluorescent protein. Analytical Biochemistry, 611, 113936. https://doi.org/10.1016/j.ab.2020.113936

Chui, C. S. L., Cowling, B. J., Lim, W. W., Hui, C. K. M., Chan, E. W., Wong, I. C. K., & Wu, P. (2020). Patterns of inpatient antibiotic use among public hospitals in Hong Kong from 2000 to 2015. Drug Safety, 43(6), 595–606. https://doi.org/10.1007/s40264-020-00920-9

Chung, S. S., Zheng, J. S., Burket, S. R., & Brooks, B. W. (2018). Select antibiotics in leachate from closed and active landfills exceed thresholds for antibiotic resistance development. Environment International, 115, 89–96. https://doi.org/10.1016/j.envint.2018.03.014

Economist Impact. (2024, September). Sea sickness: The pharmaceutical pollution problem. Back to Blue Initiative. https://backtoblueinitiative.com/sea-sickness-the-pharmaceutical-pollution-problem/

Ivantsova, N. A., Kuzin, E. N., Kuznetsov, V. V., Filatova, E. A., Pirogov, A. V., Timchenko, Y. V., Poineau, F., German, K. E., Averina, Y. M., & Kolesnikov, A. V. (2025). Advanced electrochemical treatment of tetracycline‑contaminated water using Pt/Ti electrodes. RSC Advances, 15(27), 21785–21793. https://doi.org/10.1039/d5ra02632f

Leung, H. W., Jin, L., Wei, S., Tsui, M. M., Zhou, B., Jiao, L., Cheung, P. C., Chun, Y. K., Murphy, M. B., & Lam, P. K. (2013). Pharmaceuticals in tap water: Human health risk assessment and proposed monitoring framework in China. Environmental Health Perspectives, 121(7), 839–846. https://doi.org/10.1289/ehp.1206244

Massana Roquero, D., & Katz, E. (2022). “Smart” alginate hydrogels in biosensing, bioactuation and biocomputing: State‑of‑the‑art and perspectives. Sensors and Actuators Reports, 4, 100095. https://doi.org/10.1016/j.snr.2022.100095

Minh, T. B., Leung, H. W., Loi, I. H., Chan, W. H., So, M. K., Mao, J. Q., Choi, D., Lam, J. C., Zheng, G., Martin, M., Lee, J. H., Lam, P. K., & Richardson, B. J. (2009). Antibiotics in the Hong Kong metropolitan area: Ubiquitous distribution and fate in Victoria Harbour. Marine Pollution Bulletin, 58(7), 1052–1062. https://doi.org/10.1016/j.marpolbul.2009.02.004

Wilkinson, J. L., Boxall, A. B. A., Kolpin, D. W., Leung, K. M. Y., Lai, R. W. S., Galbán‑Malagón, C., Adell, A. D., Mondon, J., Metian, M., Marchant, R. A., … Teta, C. (2022). Pharmaceutical pollution of the world’s rivers. Proceedings of the National Academy of Sciences of the United States of America, 119(8), e2113947119. https://doi.org/10.1073/pnas.2113947119

Yang, W., Moore, I. F., Koteva, K. P., Bareich, D. C., Hughes, D. W., & Wright, G. D. (2004). TetX is a flavin‑dependent monooxygenase conferring resistance to tetracycline antibiotics. The Journal of Biological Chemistry, 279(50), 52346–52352. https://doi.org/10.1074/jbc.M409573200