Table of Contents
Outline/Intro.
When discussing bioengineering and its related concepts such as synthetic biology and genetic engineering, compared to natural biological systems, the uncertainty and unpredictability of artificial systems require the introduction of engineering concepts into life system-related experiments before we can successfully achieve our goals. This is where the Design, Build, Test, Learn (DBTL) cycle comes in. With this concept, when errors occur in our experiments or designs, it provides us with a clear process to establish a new cycle, guiding our bioengineering system toward gradual improvement.
I. Establishing growth models for E. coli and Lactococcus lactis MG1363 (probiotic)
First cycle: Initial Understanding and Instrument Limitation
Design：Initially, we plotted OD600 against time to understand the concentration changes in bacterial strains over time, which allowed us to determine the right moment for CFU counting.
Build：We cultured the bacterial suspension at an appropriate temperature until the OD600 reached approximately 0.5, then performed a 100-fold dilution to prepare for the experiment.
Test：Based on the OD600-time test, we conducted the CFU-time experiment and plotted the results.
Learn：We found that the stationary phase occurred much later than expected. After discussion, we concluded that the apparent stabilization was not due to the bacteria entering the stationary phase but was likely caused by the machine reaching its detection limit. Additionally, despite extending the experiment over a longer time frame than the reference literature, we were still unable to detect the decline phase. After consulting with our professor, we learned that literature should serve as a guideline, not a strict rule.
Second cycle: Refining the CFU-Time Curve
Design：After identifying the error in our initial test, we decided to complete the CFU-time curve as a second version of the pretest.
Build：We prepared a new bacterial suspension in the same way as the previous attempt.
Test：Based on the previous results, we created a more accurate and complete CFU-time curve.
Learn：From the final experiment, we successfully obtained a clear curve. Through these two cycles, we learned the importance of validating results when data approaches the limits of the instrument.
II. Bacterial growth after adding AMP
First cycle: AMP Treatment and Its Effects on E. coli (DH5α)
Design：To evaluate the bactericidal and inhibitory effects of AMP on E. coli (DH5α), we treated the bacteria with different concentrations of AMP to observe dose-dependent effects over time.
Build：Bacterial cultures were prepared similarly to how we plotted growth curves, with AMP concentrations of 0, 10, 25, 50, 100, and 250 μg/mL, incubated at 37°C with shaking.
Test：CFU results were recorded at 0 and 24 hours.
Learn：We observed that certain concentrations of AMP stimulated E. coli growth or caused only minor changes. However, microscopy revealed bacterial debris compared to the control, indicating bacterial lysis. We hypothesized this discrepancy was due to bacterial concentration and time scale.
Second cycle: Verification of Concentration and Time Scale Effects
Design：To test whether bacterial concentration and time scale were influencing the results, we referenced other studies to determine the precise period in which AMP acts.
Build：Using the previously established growth curve function and DL modeling, we back-calculated the bacterial concentration from OD values to prepare cultures at specific concentrations.
Test：E. coli in the exponential phase (5 × 10^5 cells/mL) was treated with AMP, and frequent measurements were performed over a 4-hour period, with 24-hour results as reference.
Learn：A gradual decrease in bacterial concentration occurred within the first 4 hours, and the growth curve showed a rightward shift due to AMP treatment over a longer time scale.
III. Plasmid transformation
First cycle：Single Plasmid Transformation into DH5α
Design：In this phase, you aimed to test whether single plasmid transformation could be achieved using different plasmids in DH5α cells.
Build：You prepared the competent cells and mixed them with plasmids for transformation following standard protocols.
Test：The transformation samples were incubated with selective antibiotics, and DNA electrophoresis was used to confirm transformation.
Learn：The experiment successfully demonstrated that single plasmid transformation in DH5α was achieved.
Second cycle：Double Plasmid Transformation in DH5α
Design：You sought to enhance protein expression efficiency by performing a dual plasmid transformation into DH5α, starting with pKJE7 and then introducing the second plasmid.
Build：The chaperone plasmid pKJE7 was transformed first, followed by a second plasmid containing rhGM-CSF, as instructed by the manufacturer’s COA.
Test：Double antibiotic selection was applied, and plates were checked for colony formation. Limited colonies formed, and electrophoresis confirmed that transformation was successful in some cases.
Learn：You concluded that DH5α can harbor dual plasmids but with a low success rate, prompting you to attempt transformation in BL21 for improved expression.
Third cycle：Double Plasmid Transformation in BL21
Design：To prepare for protein expression, you planned to transform the plasmids sequentially into BL21 using the standard transformation protocol.
Build：The same transformation procedure was followed, and the incubation time was extended based on previous experience to improve success.
Test：After applying double antibiotic selection, no colonies formed, and OD600 showed no significant change. Repeated attempts confirmed the failure of transformation.
Learn：pKJE7 successfully transformed into BL21, but it became apparent that BL21 may only tolerate a single plasmid transformation.
Forth cycle：Single-Step Dual Plasmid Transformation in BL21
Design：Based on the previous hypothesis, we modified the transformation protocol to merge the two plasmid transformations into a single-step procedure, aiming to achieve a strain of BL21 successfully transformed with both plasmids in one operation.
Build：We adjusted the transformation mixture by using a ratio of 50 μL of competent cells: 10 μL plasmid 1: 10 μL plasmid 2, while following the standard transformation protocol in all other steps, allowing BL21 to take up both plasmids simultaneously.
Test：The transformed samples were cultured in medium containing both antibiotics for selection, and then subjected to electrophoresis for verification.
Learn：The OD600 of the double antibiotic-resistant culture showed an upward trend after incubation, and electrophoresis confirmed the presence of both plasmids. This validated our previous cycle’s hypothesis and successfully produced a BL21 strain containing both plasmids, which was preserved for future protein expression efficiency experiments.
IV. The preparation of the hydrogel for probiotic
First cycle: Selection of the Ideal Hydrogel Protocol
Design: The goal was to conduct hydrogel formation experiments without altering the pH to identify the optimal hydrogel formulation. Different ratios of chitosan solution and 1% glutaraldehyde were tested, followed by qualitative analysis, including compression testing, to determine the required ratio.
Build: Ten different hydrogel formulations with varying chitosan/glutaraldehyde ratios were prepared, ranging from 5/0.5 to 5/5.0. These formulations were then subjected to qualitative tests based on properties such as viscosity, elasticity, adhesiveness, and resistance to compression.
（Chitosan solution / 1% glutaraldehyde）
| 1 | 2 | 3 | 4 | 5 |
| 5/0.5 | 5/1.0 | 5/1.5 | 5/2.0 | 5/2.5 |
| 6 | 7 | 8 | 9 | 10 |
| 5/3.0 | 5/3.5 | 5/4.0 | 5/4.5 | 5/5.0 |
note： Formula No. 6 is based on the formulation used in the referenced paper.
Test: Each formulation was evaluated for its ability to form a gel, adhere to the skin, maintain elasticity, and resist breaking under pressure. Formulations were compared based on their respective properties, such as viscosity, elasticity, and the ability to stay attached to the skin.
Learn: Among the tested formulations, Sample No. 2 (5:1 ratio) was determined to be the most suitable hydrogel for the application. This formulation provided a good balance of adhesion, elasticity, and moisture retention while maintaining structural integrity and avoiding detachment from the skin.
| No. | Properties |
| 1 | Does not form a gel |
| 2 | High viscosity, adheres well to the skin without falling off, elastic |
| 3 | Reduced viscosity and elasticity (compared to 2), but still adhesive, adheres to the skin without falling off |
| 4 | More solid, less elastic, slightly brittle but acceptable, adheres to the skin without falling off, feels crumbly under pressure |
| 5 | Lower viscosity, doesn’t stick much to cling film, adheres to the skin without falling off, feels crumbly under pressure, does not break apart when picked up with tweezers |
| 6 | Similar to 5 |
| 7 | Greater resistance when pressed (compared to 5 and 6), more compact and elastic |
| 8 | Sticky, strong rebound when pressed (8 > 9 > 10), compact and elastic |
| 9 | Falls off the skin, strong rebound when pressed (8 > 9 > 10), compact and elastic |
| 10 | Falls off the skin, strong rebound when pressed (8 > 9 > 10) |
Second cycle: pH Adjustment for Probiotic Growth
Design：The objective was to adjust the pH of the growth medium to match optimal conditions for Lactococcus lactis, specifically aiming for a pH range of 6.3 to 6.5.
Build：NaOH was used to raise the pH, and the hydrogel properties were analyzed using the same ratios as in previous experiments.
Test：During pH adjustment, the solubility and stability of chitosan were monitored. White precipitates formed as pH increased, and centrifugation was performed to separate the clearer chitosan solution.
Learn：The rise in pH affected the solubility of chitosan, leading to the formation of aggregates, which required additional steps such as centrifugation for clearer solutions.
Third cycle: Probiotic Growth Testing Methods
Design: The goal was to test the suitability of the hydrogel for probiotic bacterial culture using three methods: injection, plate coating, and mixing. Each method aimed to evaluate bacterial growth and hydrogel stability under different conditions.
Build: Three distinct approaches were employed. In the injection method, 5 µL of bacterial culture was injected into a preformed hydrogel. The mixing method involved thoroughly blending the bacterial culture with the chitosan solution before adding the crosslinking agent. The plate coating method mixed MRS broth with chitosan solution, followed by spreading on a plate and adding bacterial culture.
| Injection Method | Using the selected ratio (5 mL chitosan solution mixed with 1 mL glutaraldehyde), the hydrogel was allowed to form. We then injected 5 µL of bacterial culture into the gel using a pipette tip. |
| Mixing Method | The bacterial culture was mixed thoroughly with the chitosan solution before adding the crosslinking agent. |
| Plate Coating Method | MRS broth was mixed with the chitosan solution and spread on a plate, followed by the addition of the crosslinking agent. We then added 50 µL of bacterial culture to the plate. |
Test: Bacterial viability was measured using OD readings for the injection and mixing methods. However, water release led to a phase separation, causing inconsistent OD readings due to uneven light refraction. The plate coating method resulted in clumps due to the MRS broth preventing gel formation, rendering it unusable.
Learn: The increase in pH caused the hydrogel to become more rigid and less elastic, particularly affecting Sample No. 1, which failed to form a gel. The injection and mixing methods suffered from water release issues, compromising OD measurement reliability, while the plate coating method proved ineffective for gel formation.
Forth cycle: Modified Plate Coating Method
Design：After initial failures, the plate coating method was revised based on literature recommendations to better support bacterial growth.
Build：Chitosan hydrogel was allowed to fully form on the plate, followed by the addition of MRS broth, which was incubated for 12-16 hours. The broth was then removed, and bacterial culture was spread over the hydrogel.
Test：The hydrogel surface became uneven after the addition of MRS broth, but bacterial colonies were visible after 2-3 days of incubation.
Learn：Despite surface irregularities, the probiotics were able to grow and form visible colonies, indicating that the hydrogel matrix supported bacterial survival and growth.
V. Cell culture for growth factor activity test
First cycle: Thraw Cell and Cell Culture
Design：The cells used in this experiment were TF-1 cells. After ordering the cells from a supplier, they were thawed from liquid nitrogen and cultured to restore their healthy state for the subsequent experiments.
Build：The growth environment required for the cells was first prepared, including the necessary medium and nutrients. The cells were then thawed, centrifuged, and resuspended in the prepared medium, followed by incubation for 2-3 days.
Test：After approximately 3 days of culture, with regular medium changes and passaging, the medium turned yellow and appeared grainy, and there were few healthy adherent cells, leading to the suspicion that the cells were contaminated.
Learn：When culturing cells, a small amount of antibiotics is usually added to the medium to prevent contamination. This is especially important when the cells are freshly thawed, as they are more fragile and require extra care during cultivation.
Second cycle: Medium Contaminated Confirm
Design：To determine whether contamination occurred during handling or if the medium itself contained bacteria or impurities, the medium was cultured separately in a test tube to observe for contamination.
Build：The cell culture medium was placed into a test tube and incubated overnight, observing for changes in turbidity and color.
Test：The results showed that the original medium remained clear, suggesting the medium itself was not contaminated. However, the medium that had been aliquoted became noticeably turbid after overnight incubation, indicating bacterial contamination.
Learn：Improper handling during the process led to contamination of the medium during cell culture, resulting in poor cell condition.
Third cycle: Antibiotic Test
Design：After the previous contamination, we thawed a new vial of cells and cultured them with a small amount of antibiotics, paying extra attention to handling techniques to prevent contamination.
Build：While preparing the medium, 1% ampicillin was added. The cells were then thawed, centrifuged, and resuspended in the prepared medium for sub/culturing and passaging.
Test：The medium remained clear, and under the microscope, the cells appeared to be in a healthier state.
Learn：Greater caution should be exercised during cell culture handling, and adding appropriate antibiotics to the environment can help inhibit bacterial growth.
