Measurement

Introduction

In synthetic biology, functional assays are one of the most critical experimental steps, consisting of sample preparation, control setup, measurement execution, data collection, and result analysis. Traditionally, during sample preparation, enzymes are produced using LB + IPTG induction. However, this IPTG-based approach requires manual addition of the inducer when the culture reaches an optical density (OD) of 0.6–0.8. This not only increases the likelihood of random human errors but also makes high-throughput testing difficult to achieve.

To overcome these limitations, we developed a high-throughput measurement system for characterizing protein variants generated by mutagenesis, centered on ZYM-5052 auto-induction medium. This system enables automatic protein expression without manual intervention, reducing inconsistencies while greatly improving efficiency and scalability, thereby allowing us to characterize mutant variants more rapidly and reliably.

The concept of auto-induction was first introduced by F. William Studier in 2005, who demonstrated that under appropriate metabolic conditions, E. coli can automatically initiate protein expression and achieve yields typically several-fold higher than conventional IPTG induction. As Studier summarized: “Auto-induction allows efficient screening of many clones in parallel for expression solubility, as cultures have only to be inoculated and grown to saturation, and yields of target protein are typically several-fold higher than obtained by conventional IPTG induction.” Among the various auto-induction medium developed (such as SILEX, QS-based, and pH/oxygen-based systems), we chose ZYM-5052 for its stability, maturity, and reliability, making it the foundation of our optimized functional assay.

Advantages of Our System

As mentioned previously, ZYM-5052 enables consistently high levels of protein expression, ensuring that mutant proteins reach sufficient expression for measurement is needed. This reduces variability caused by expression differences and allows us to focus on detecting the true functional differences in enzyme activity between variants. Moreover, the system offers two main advantages. First, its compatibility with automated procedures allows for the simultaneous processing of a large number of mutant proteins. Second, due to the auto-induction properties of the medium itself, it improves both the efficiency and reproducibility of the assays.

Principle of ZYM-5052

In T7 system, a repressor protein (lacI) binds to the operator region, preventing T7 RNA polymerase from transcribing downstream genes, thereby temporarily suppressing the expression of the target protein. IPTG exploits this feature and serves as an artificial inducer to control gene expression. When IPTG is present, it binds to lacI, causing the repressor to dissociate from the operator, lifting the transcriptional block and allowing the gene to be transcribed and expressed as protein. IPTG is a lactose analog that is not metabolized by the cell, making it a stable and reliable inducer.

The mechanism of ZYM-5052 medium is similar to IPTG but operates in a more natural, auto-regulated manner. ZYM-5052 is a medium containing a mixture of glucose, glycerol, lactose, and trace elements, designed for auto-induction. In this medium, lactose gradually becomes available as an inducer once glucose is depleted when E. coli grows to the mid-late log phase , binds to lacI, removes the repressor from the operator, and initiates protein expression.

Moreover, high concentrations of glucose suppress the expression of the lactose operon through the cAMP/CAP system: when glucose is abundant, intracellular cAMP levels are low, preventing the cAMP-CAP complex from binding to the promoter and inhibiting the initiation of the lactose operon. This glucose-dependent repression is a key mechanism underlying ZYM-5052 auto-induction. Unlike IPTG, which triggers immediate induction upon addition, induction in ZYM-5052 occurs only after glucose is nearly exhausted and cell density is high. This allows protein expression to coincide naturally with cell growth, initiating expression when nutrient utilization is maximized and the culture has reached high density, reducing the metabolic burden during early exponential growth.

In summary, IPTG provides controllable and prompt artificial induction, whereas ZYM-5052 leverages glucose depletion and lactose auto-induction for growth-synchronized and optimized protein expression.

Comparability

In traditional protein expression systems, a common method involves adding IPTG to LB medium. While effective, this method necessitates manual addition of IPTG, which is operationally cumbersome for high-throughput characterization of a large number of variants.

To address this limitation, we designed a high-throughput measurement system based on the ZYM-5052 autoinduction medium. By eliminating the need for manual induction, ZYM-5052 allows us to process large-scale variants libraries more efficiently while also supporting stronger protein expression. Therefore, we hypothesize that ZYM-5052 is a more practical and reliable foundation for high-throughput measurement compared to the conventional LB + IPTG method.

To validate this hypothesis, we performed a direct comparison of the two systems. We selected GFP as a standardized reporter protein and included a vector-only construct as a negative control to ensure that the observed fluorescence signal genuinely represents protein expression.

Cultures were grown in either LB or ZYM-5052. For the LB group, IPTG was added when the OD value reached 0.6−0.8. Samples were collected two hours after induction to measure both the OD600 and GFP fluorescence. To ensure the accuracy of our results, we performed eight biological replicates for each method.The following is our protocol.

Reproducibility

During the comparative experiment, we followed a standardized procedure. The protocol is as follows:

ZYM-5052 Setup

I. Materials

1. Dry Component
• Tryptone: 10 g
• Yeast Extract: 5 g
• Deionized water: ~958 mL (leave space for adding stocks)
2. Stock Solutions (prepare in advance under sterile conditions)
• 50× M Stock (phosphate and nitrogen source)
• Na₂HPO₄·7H₂O: 335 g/L
• KH₂PO₄: 170 g/L
• NH₄Cl: 134 g/L
• Na₂SO₄: 35.5 g/L
• Preparation: dissolve salts in ~640 mL water, mix thoroughly, sterilize, and store at room temperature or 4 °C.
• 50× 5052 Stock (carbon source mixture: glycerol/glucose/α-lactose)
• Glycerol: 25% w/v (250 g/L)
• Glucose: 2.5% w/v (25 g/L)
• α-Lactose: 10% w/v (100 g/L)
• Preparation: dissolve lactose first (may require gentle heating or long stirring), then add glycerol and glucose. Filter sterilize (0.22 μm) or autoclave if compatible.
• 1 M MgSO₄
• MgSO₄·7H₂O: 24.65 g / 100 mL
• Sterilize and store at 4 °C.
• 1000× Trace Elements
• Prepare according to UNC recipe with appropriate metal ions dissolved in acidic solution (HCl).
• Sterilize and store at 4 °C.
3. Antibiotics (if required)
• Kanamycin: 100 µg/mL (adjust according to plasmid and experimental requirements)

II. Procedure

1. Step 1: Dissolve Dry Components
1. Add ~958 mL deionized water to a graduated cylinder or suitable vessel.
2. Add 10 g Tryptone and 5 g Yeast Extract.
3. Stir thoroughly using a magnetic stir bar or stir rod until fully dissolved.
2. Step 2: Sterilization
1. Transfer solution to an autoclavable container.
2. Autoclave at 121 °C for 15–20 minutes.
3. Cool to room temperature or ~50 °C.
3. Step 3: Add Sterile Stock Solutions
1. Under sterile conditions, add:
• 50× M stock: 20 mL
• 50× 5052 stock: 20 mL
• 1 M MgSO₄: 2 mL
• 1000× Trace Elements: 0.2 mL
• Antibiotic (if required)
2. Mix thoroughly using a magnetic stirrer or stir rod.
3. Adjust final volume to 1 L with deionized water.
4. Step 4: Check and Store
1. (Optional) Dilute 1:10 to measure pH, approximately 6.7.
2. Use immediately for bacterial inoculation and auto-induction expression.
3. For temporary storage: keep under sterile conditions at 4 °C and use as soon as possible.

LB Setup

I. Materials

• LB Broth powder
• Source: Difco™ LB Broth, Miller (BD 244620) or Sigma–Aldrich L3522
• Composition: Tryptone, Yeast Extract, NaCl (pre-mixed)
• Deionized / Distilled water (dH₂O)
• Laboratory-grade purity
• Antibiotic stock solutions
• Ampicillin 100 mg/mL or Kanamycin 50 mg/mL (we used the latter one)
• Storage: −20 °C
• Autoclavable Erlenmeyer flask or medium bottle
• Leave ≥20 % headspace before autoclaving
• Autoclave
• Set to 121 °C for 15–20 min

II. Procedure

• Liquid LB Broth (1L)
1. Weigh 25 g Difco™ LB Broth (Miller) powder (or equivalent brand).
2. Add to ~800 mL dH₂O in an autoclavable bottle or flask.
3. Stir until fully dissolved, then bring volume to 1 L with dH₂O.
4. Cap loosely and autoclave at 121 °C for 15–20 min.
5. Allow to cool to ~50 °C.
6. Add antibiotic (if required):
• Ampicillin 100 µg/mL → add 1 mL of 100 mg/mL stock per 1 L.
• Kanamycin 50 µg/mL → add 1 mL of 50 mg/mL stock per 1 L.
7. Mix gently, label, and store at 4 °C (stable 2–3 weeks).

Sample Preparation

1. Prepare 3 ml of LB medium and 3 ml of ZYM-5052 medium in separate 50-ml conical tubes.
2. After overnight growth in 6 ml LB culture, transfer 30 µL of culture into two separate 50‑ml conical tubes, which were prepared in step 1.
3. For the LB medium group, grow the E. coli culture until the OD600​ reaches 0.6−0.8. Then, induce GFP expression by adding IPTG to a final concentration of 0.3 mM.
4. Incubate the cultures at 37 °C for 2 hours before screening. Wait for the expressing of GFP/pET-29a

Screening

1. After 2 hours of incubation, distribute 100 µL of the cultures to a 24-well plate
2. Measure OD600 (for cell density) and GFP fluorescence (excitation 418 nm / emission 513 nm) in a microplate reader, taking readings at 1-minute intervals.

Reliability

When culturing bacteria in LB medium, IPTG had to be added once the OD reached 0.6–0.8. However, as shown in Figure 2 and 3, after three hours of GFP strain cultivation in LB medium, the OD values varied noticeably between groups.

We suspect this variation resulted from pipetting errors or slight differences in the timing of IPTG addition, which led to asynchronous experiments across replicates. In contrast, this problem did not occur with ZYM-5052, since no manual induction is required. Once glucose in the medium is depleted, lactose derivatives bind to LacI, remove the repressor from the operator, and automatically initiate protein expression. This mechanism avoids pipetting errors and timing differences that would otherwise compromise synchronization.

Because protein expression levels are influenced by bacterial growth, we also compared the average peak OD600 values obtained under the two systems (Figure 4 & 5 ).

The results showed that whether using vector-only or GFP strains, cultures grown in ZYM-5052 reached significantly higher cell densities than those in LB. Even though the error range in ZYM-5052 cultures was slightly broader, it did not overlap with that of LB, clearly indicating that ZYM-5052 supports much higher biomass accumulation. This directly translates into higher protein yields, as confirmed by subsequent measurements.

Next, we compared the effects of the two methods on protein expression.

Protein expression data further validated the superiority of ZYM-5052 for high-throughput applications. As shown in Figure 6, the vector-only control showed no detectable GFP expression, while both LB+IPTG and ZYM-5052 groups produced strong signals with minimal variation between replicates. During the first five hours, LB+IPTG cultures exhibited higher GFP levels, likely because ZYM-5052 cultures had not yet reached peak density. However, when comparing the OD growth trends (Figure 7) with protein expression trends (Figure 6), we observed that maximum protein expression coincided with the peak in cell density. At this stage, ZYM-5052 significantly outperformed LB+IPTG, yielding several-fold higher protein expression levels.

Visual inspection of cultures further supported these findings, as the GFP signal in ZYM-5052-grown(GZ1-8) cultures was much stronger than in those induced with IPTG(GI1-8).

In conclusion, while IPTG induction offers a slightly faster onset of expression, ZYM-5052 consistently delivers higher yields and greater scalability, making it especially advantageous for high-throughput screening of enzyme variants. Both systems proved stable and reproducible, but the stronger performance of ZYM-5052 ultimately led us to adopt it as the foundation of our measurement strategy. We believe that this approach does not only enhance the efficiency of our project but may also serve as a valuable reference for future iGEM teams seeking reliable, scalable methods for protein measurement.

Scalability

Application of PLA-degrading enzyme variants

This year, our project aimed to generate a large library of variant enzyme-expressing genes (For details, please see the engineering page.), during which we employed the high-throughput measurement system we developed.The following are the steps of our system and the results of the experiment.

Mutagenesis

First, because we needed multiple variants, we used Mutazyme II DNA polymerase to perform error-prone PCR to obtain mutant genes. These mutant genes were first cloned into the plasmid and then transformed into E. coli DH5α as an intermediate host, and finally into E. coli BL21 for protein expression.

Of course, other mutagenesis methods could also be applied; whenever there is a need for large-scale screening, our approach can be used to conveniently express the proteins.

Auto-induction

Perform auto-induction by using the same protocol of ZYM-5052 Setup and described above for the Reproducibility.

Cell Lysis

Furthermore, we extracted crude proteins for subsequent assays. After approximately 16 to 20 hours of cultivation, proteins had accumulated abundantly inside the cells, at which point the cultures were harvested by low-temperature centrifugation. The collected cells were then roughly lysed using 0.1 mm glass beads in combination with vortex mixing.

1. Take 1400 μL of culture medium and transfer it into a centrifuge tube.
2. Centrifuge and discard the supernatant.
3. Add 800 μL of TRIS buffer to wash the pellet, then transfer it into an Eppendorf tube.
4. Centrifuge again, remove the supernatant, and resuspend the pellet with 200 μL of TRIS buffer by pipetting until fully dissolved.
5. Perform another centrifugation and discard the supernatant.
6. Add 200 μL of TRIS buffer and introduce 0.1 mm glass beads (up to the 0.1 mark of the Eppendorf tube).
7. Vortex the tube.
8. Centrifuge once more to prevent glass beads from being aspirated.
9. Keep 20 μL of the supernatant on ice until use.

Alternatively, other extraction methods could also be employed. As long as the approach meets the requirement for parallel testing of a large number of variants, the use of ZYM-5052 medium allows each variant to automatically express proteins, thereby reducing manual handling, uncertainty, and time consumption.

pNPB assay

Finally, for the measurement step, the crude protein extracts were aliquoted into 24-well plates, and enzymatic activity was assessed using pNPB as the substrate for screening. To elaborate, the pNPB assay is a colorimetric method designed to measure the activity of lipases or esterases. The enzyme hydrolyzes pNPB into p-nitrophenol (pNP), which appears yellow under alkaline conditions and can be measured at a wavelength of 405 nm. The rate of absorbance increase is proportional to enzyme activity, allowing the determination of enzyme activity levels, with OD used as a comparative indicator.

• Pre-assay preparation
  • Collect enzyme-containing supernatants after cell lysis.
  • Keep samples on ice to maintain stability until use.
• Materials required
  • pNPB stock solution
  • Tris buffer
  • 24-well plate or cuvette
  • Microplate reader or spectrophotometer (λ = 405 nm)
• Preparation of pNPB stock solution
  • Weigh an appropriate amount of solid pNPB.
  • Dissolve in anhydrous isopropanol or ethanol to obtain a 10–20 mM stock solution.
  • Store in light-protected microtubes at –20 °C to prevent photolysis and spontaneous hydrolysis.
  • Before use, dilute with Tris buffer to the required final concentration.
• Measurement procedure
  1. Mix 20 μL enzyme sample with 100 μL Tris buffer.
  2. Add an appropriate amount of diluted pNPB stock solution to initiate the reaction.
  3. Immediately measure absorbance at 405 nm, recording data every 1 minute for 5–10 minutes.
• Controls
  • Blank control: Tris buffer + pNPB only (to correct spontaneous hydrolysis).
  • Negative control: bacterium not expressing the enzyme (to confirm the activity source).
• Note
  • The entire assay should avoid light exposure and prevent spontaneous hydrolysis of the reagents.

Results

We ran the pNPB assay. The results are in Figure 10-13.

The results of the pNPB assay showed that after comparing the mutant variants to the GFP and the wild type; This system enabled us to rapidly screen out superior variants, thereby facilitating the progress of our project.

Conclusion

Under conditions requiring high-throughput put testing, ZYM-5052 shows significantly better synchronization compared to the conventional IPTG method. Therefore, we developed ZYM-5052 into a High-throughput Measurement System(As shown in Figure 13), which enables efficient protein expression through four steps: gene mutation, auto-induction, protein extraction, and screening. This system not only offers ease of operation and stability but also contributes to the success of our engineering goals. Moreover, we believe that this approach accelerates the experimental workflow while maintaining stability and accuracy, making it a feasible and reliable strategy.

In addition, its applications are not limited to mutation, GFP assays, or pNPB assays. Other substrates may also be employed depending on the target activity to be tested. Our central idea is to cultivate different mutant variants individually in ZYM-5052 medium, allowing them to automatically express proteins, and then evaluate the functional properties of these expressed proteins.