This section presents the successful construction and functional validation of the three core modules designed for our "ProBabyotics" platform, aimed at systemically enhancing the nutritional value of infant formula. Each module was systematically engineered and rigorously tested, providing a robust proof-of-concept for our overall project.
Collectively, these results validate the feasibility of our modular approach. We have successfully produced and characterized all three key functional components, laying a solid scientific foundation for the development of our next-generation infant formula, "2'F-Luxe".
Figure 1: ProBabyotics: Engineering a Better Formula
This section details the systematic engineering of an E. coli strain for the de novo biosynthesis of 2'-fucosyllactose (2'-FL), a key human milk oligosaccharide(Yang et al., 2025). Our project followed a multi-round 'Design-Build-Test-Learn' (DBTL) cycle to progressively identify and overcome metabolic bottlenecks(Zhao et al., 2025)a. Key strategies included constructing a complete five-gene metabolic pathway, optimizing the host chassis to eliminate substrate competition, and employing protein engineering to enhance the activity of a rate-limiting enzyme. Through these iterative improvements, we successfully increased the 2'-FL titer from an initial proof-of-concept yield of 8.10 mg/L to a final yield of
The objective of this experiment was to provide a
Synthesize manB and manC and clone the pET28a vector through NdeI and XhoI restriction sites. E. coli BL21 (DE3) (D1009S) and DH5α (DE3) (D0351) supercompetent cells were purchased from Beyotime. Subsequently, the recombinant plasmid is transformed into E. coli DH5α and BL21. Positive clones are screened on LB (Luria Bertani) plates containing 100 μg/mL kanamycin (Kana), followed by sequencing verification (Beijing, Jingke) to obtain the recombinant engineering strain. The strain is stored at -80°C with 25% (v/v) glycerol as a cryoprotectant.
Figure 2: Construction of BL21-manB+manC. (A) The plasmid map of pET28a-manB+manC; (B) The gene circuit of BL21-manB+manC; (C) Flow chart of Genetic engineering
Transfer the recombinant strains in 50mL Erlenmeyer flasks containing 10 mL LB medium with Kana, under conditions of 25°C, 250 rpm. Measure cell concentration by optical density at 600 nm using a microplate reader （FlexStation 3，Molecular Devices，USA）. When OD₆₀₀ reaches 0.6, bacteria are collected by centrifugation at 4000 g for 5 min and resuspended into 100 mL M9 Broth (1 mM MgSO₄ and 50 μM CaCl₂)(A510881, Sangon Biotech, China) with 20 g/L glycerol and 5 g/L yeast extract. As cell OD₆₀₀ reached 0.6 again , 0.5 mM IPTG and 8 g/L lactose are added to produce 2'-fucosyllactose (2'-FL).
Figure 3: Flow chart of bacterial culture.
To quantify the 2'-FL concentration, 1 mL of fermented broth was collected at designated intervals. The samples were centrifuged at 10,000 g for 1 minute, and the resulting supernatant was collected and stored at -20°C for subsequent analysis.
The 2'-FL content was determined using a commercial detection kit (Synome, 680112), which relies on a two-step enzymatic cascade. A highly specific α-L-fucosidase first hydrolyzes 2'-FL to release L-fucose. Subsequently, L-fucose dehydrogenase (FDH) oxidizes the L-fucose to L-fucono-1,5-lactone. This reaction is coupled with the stoichiometric reduction of NADP⁺ to NADPH. Since NADPH has a strong characteristic absorbance at 340 nm, the increase in absorbance is directly proportional to the amount of NADPH produced, which is equimolar to the initial amount of 2'-FL in the sample. The final concentration was calculated based on the change in absorbance at 340 nm (A₃₄₀).
Figure 4: Flow chart of 2'-FL content assay
The results of the agarose gel electrophoresis are shown in Figure 5. A distinct band of the expected size (1947 bp) was observed, confirming the successful express of manB+manC.
Figure 5: the agarose gel electrophoresis analysis of the manB+manC gene fragment
The 2'-fucosyllactose content in both BL21 and BL21-p28a is close to 0 mg/L, while the average 2'-fucosyllactose content of BL21- manB+manC is 8.10±1.03 mg/L. 2'-fucosyllactose content is higher in BL21-manB+manC than in BL21 and BL21-p28a.
Figure 6: Comparison of 2'-FL yield in BL21-manB+manC after 72-hour fermentation
The results demonstrate that the co-expression of manB and manC is
This experiment aims to evaluate the effect of expression of different genes in BL21 recombinant strains on the yield of 2'-FL after 72 hours of fermentation.
Synthesize manB, manC, gmd，fcl and futC, and Clone the pET28a vector through NdeI and XhoI restriction sites. E. coli BL21 (DE3) (D1009S) and DH5α (DE3) (D0351) supercompetent cells were purchased from Beyotime. Subsequently, the recombinant plasmid is transformed into E. coli DH5α and BL21. Positive clones are screened on LB (Luria Bertani) plates containing 100 μg/mL kanamycin (Kana), followed by sequencing verification (Beijing, Jingke) to obtain the recombinant engineering strain. The strain is stored at -80°C with 25% (v/v) glycerol as a cryoprotectant.
Figure 7: Construction of BL21-manB+manC+gmd+fcl+futC. (A) The plasmid map of pET28a-manB+manC+gmd+fcl+futC (B) The gene circuit of BL21-manB+manC+gmd+fcl+futC.
Transfer the recombinant strains in 50mL Erlenmeyer flasks containing 10 mL LB medium with Kana, under conditions of 25°C, 250 rpm. Measure cell concentration by optical density at 600 nm using a microplate reader （FlexStation 3，Molecular Devices，USA）. When OD₆₀₀ reaches 0.6, bacteria are collected by centrifugation at 4000 g for 5 min and resuspended into 100 mL M9 Broth (1 mM MgSO₄ and 50 μM CaCl₂)(A510881, Sangon Biotech, China) with 20 g/L glycerol and 5 g/L yeast extract. As cell OD₆₀₀ reached 0.6 again , 0.5 mM IPTG and 8 g/L lactose are added to produce 2'-fucosyllactose (2'-FL).
Figure 8: Flow chart of bacterial culture.
Collect 1mL fermented broth from cultured bacteria at intervals. After centrifugation at 10,000g for 1 min, the supernatant is stored at -20°C. Calculate the 2'-FL content according to the instruction(680112, Synome).
Figure 9: Flow chart of 2'-FL content assay
The agarose gel electrophoresis results for the manA, gmd, fcl and futC gene are shown in Figure 10. As indicated by the marker, bands of the correct corresponding lengths were observed for all genes, confirming their successful expression.
Figure 10: Agarose gel electrophoresis analysis of the manA, gmd, fcl, futC gene fragment
We synthesise manA, manB, manC, gmd，fcl and futC at first, but due to the significantly slow growth rate of the recombinant strain after expressing manA, a literature review was conducted. It was determined that the manA gene is not essential for the 2'-FL production pathway. Therefore, the recombinant strain used for subsequent 2'-FL yield measurements contained the gene sequence manB, manC , gmd, fcl, and futC.
As shown in Figure 11, the results indicate that the 2'-FL content in both BL21 and BL21-p28a is close to 0 mg/L. And the average 2'-FL content of BL21-manB+manC and BL21-manB+manC+gmd+fcl+futC are 8.11±1.04 mg/L and 19.66±2.62 mg/L respectively. Overall, BL21-manB+manC+gmd+fcl+futC yields the most 2'-fucosyllactose.
Figure 11: Yield comparison of 2 '-fucosyllactose after 72 hours of fermentation of BL21 recombinant strain
The maximum yield of the recombinant strain BL21-manB+manC+gmd+fcl+futC reaches 19.66±2.62 mg/L of 2-FL. This indicates that overexpression of manB, manC, gmd, fcl and futC can significantly increase the yield of 2 '-FL, which is consistent with other findings(Liang et al., 2025).
Building upon our initial proof-of-concept, this experiment aimed to
The BL21-manB+manC+gmd+fcl+futC engineered strain we used is manufactured in the previous steps.
Measure cell optical density at 600nm using a microplate reader （FlexStation 3，Molecular Devices，USA). During fermentation, 1 mL broth samples are collected at designated time intervals to monitor cell growth. The bacterial cells are immediately pelleted by centrifugation at 10,000 × g for 10 minutes. After carefully removing the supernatant, the cell pellets undergo a drying process in a 50°C oven for 8 hours to completely evaporate residual moisture. The resulting dried biomass is then precisely weighed to determine the dry cell weight (DCW), as
DCW(g/L)=(Wtube+cell−Wtube) /Vsample×dilution factor .
Collect 1mL fermented broth from cultured bacteria at intervals. After centrifugation at 10,000g for 1 min, the supernatant is stored at -20°C. Then calculate the 2'-FL content according to the instruction(680112, Synome).
Repeat the first and second steps in the process '2'-FL content assay', follow by the calculation of lactose content according to the instruction(Jonln, JL-T1072).
Figure 12: Flow charts of bacteria dry cell weight determination, 2'-FL content assay and lactose content assay.
It is clear from the graph that during the period of 72 hours, the amount of lactose fall consistently while dry cell mass and the figure for 2’-FL and the dry cell mass rised from 0.00 mg/L to about 22.71 mg/L and from 0.00 mg/L to 18.00 mg/L respectively.
Figure 13: Changes in 2'-FL yield, Lactose Content, and Cell Dry Weight Over Time in BL21-manBC+gmd+fcl+futC
The results strongly support our hypothesis. The introduction of the complete five-gene cassette (manB+manC+gmd+fcl+futC) led to a final 2'-FL yield of
Additionally, we learned that including manA in the construct severely impaired cell growth, and a literature review confirmed it was non-essential (Li et al., 2025). This finding refined our final construct design. Overall, we successfully established a functional, multi-gene pathway for 2'-FL synthesis in E. coli, laying the groundwork for further optimization.
While the enzymatic assay provided a convenient method for high-throughput quantification, its detection principle is based on the measurement of L-fucose released after hydrolysis. Consequently, the assay cannot distinguish authentic 2'-FL from other potential fucose-containing byproducts (such as other fucosylated oligosaccharides) or any pre-existing free L-fucose in the fermentation broth. This inherent lack of specificity creates potential for interference and necessitates a more rigorous confirmation of product identity.
Therefore, to provide definitive and
Figure 14: Students performed the complex HPLC analysis under the guidance of our instructors.
The recombinant strains mentioned above are supplemented into the LB medium containing Kana, under the conditions of 25°C and 250 rpm. Detect the value of OD₆₀₀ and as it reaches a0.6, transfer the strains to M9 medium (20 g/L glycerol, 5 g/L yeast extract, 1 mM MgSO4, 50 μM CaCl2). As OD₆₀₀=0.6 again, the production of 2’-FL will be induced by adding 0.5 mM IPTG and 8 g/L lactose.The production of 2'-FL by engineered bacteria will be analyzed by a high performance liquid chromatography (HPLC) system (WATERS), equipped with a WATERS ACQUITY UPLC BEH Amide (2.1×100 mm, 1.7 μm). The mobile phases are A: 80% acetonitrile and 20% ammonia (0.1%), B: 30% acetonitrile and 70% ammonia (0.1%), with flow rate of 0.3 mL/min and temperature of 45°C. According to the programmed gradient, mobile phase A decreases from 90% to 60% at 0-7 min and recoveres to 90% at 7-10 min. Mobile phase B increases from 10% to 40% at 0-7 min and decreases to 10% at 7-10 min.
Figure 15: Mechanism of HPLC
The HPLC chromatogram of the fermentation supernatant from our engineered BL21 strain is presented in Figure 16. A prominent peak was detected at a retention time of
This result provides definitive evidence that our engineered metabolic pathway is functional and that the compound being produced is, in fact, 2'-FL. The successful verification of the product's identity is a critical milestone, validating our quantitative yield measurements and confirming the success of our genetic design.
Figure 16: HPLC analysis of fermentation products of BL21
Based on the knowledge that the lacZ gene in E. coli BL21 creates a competitive pathway for our lactose substrate, we hypothesized that
Our group used the same method to engineer 2 kinds of bacteria: BL21-manB+manC+gmd+fcl+futC and DH5α-manB+manC+gmd+fcl+futC by transferring designed recombinant plasmid to competent E.coli BL21 and DH5α. The concentration of 2'-FL in the sample is detected through absorbance , using a reagent kit and a microplate reader.
Figure 17: Methods for Exploring the effect of two different strains(BL21/DH5α) on 2'FL yield. (A)Experimental Methods; (B)Experimental Flowchart
We conducted a head-to-head comparison by fermenting our engineered BL21 and DH5α strains under identical conditions. The results were definitive: the DH5α strain produced
Figure 18: The effect of two different strains(BL21/DH5α) on 2'FL production
The results provide a
The futC gene, which encodes the final and rate-limiting enzyme in our pathway, is of human origin. Heterologous expression of such proteins in E. coli often leads to misfolding and poor solubility. To address this anticipated bottleneck, we employed a
First, we used the same method described above to construct the engineered strain DH5α and overexpress the manB+manC+gmd+fcl+futC gene. Then, we introduced TrxA tag fusion into DH5α. We detect the production of 2'-FL by using the 2'-FL Detection Kit according to the manufacturer’s instructions.
Figure 19: Methods for the effect of TrxA tag fusion futC on 2 '-fucosyllactose yield. (A)Experimental Methods; (B)Experimental Flowchart; (C) The gene circuit of DH5α-manB+manC+gmd+fcl+TrxA-futC.
The results showed that fusing futC with a TrxA tag significantly boosted 2'-FL production. The engineered strain with the TrxA-futC fusion yielded
Figure 20: The effect of TrxA tag fusion futC on 2 '-fucosyllactose yield
The data conclusively demonstrates the success of our protein engineering approach. The strain expressing the
This result confirms that the poor solubility of the heterologously expressed futC was indeed a major limiting factor. By leveraging the TrxA fusion tag, we successfully increased the amount of soluble, active futC enzyme in the cell. This serves as a powerful demonstration of how targeted protein engineering can be used to overcome expression challenges and unlock the full potential of a metabolic pathway (Young & Robinson, 2014).
In summary, our team successfully executed a four-stage engineering cycle that progressively improved 2'-FL production by over
Collectively, these results provide a robust, step-by-step validation of our metabolic engineering strategy. We have not only created a high-performing production strain but also documented a clear and logical workflow that can serve as a valuable reference for future iGEM teams tackling complex pathway optimization challenges (Gao et al., 2025).
This section details the complete workflow for the production of a functional recombinant protease, trypsin, designed to improve protein digestion in infant formula (Jakobsson et al., 2000). Our engineering process encompasses the entire journey from gene to function: (1)
This experiment aimed to
The TRYP gene (Uniprot: P00761) is codon-optimized according to RFC10 standards, synthesized, and cloned into the NdeI/XhoI-digested pET-28a plasmid to construct the pET-28a-TRYP recombinant vector (Buck et al., 1962). This vector is then transformed into E. coli BL21(DE3) competent cells (Beyotime, D1009S). Positive transformants are selected on LB agar plates supplemented with 100 μg/mL Kana and subsequently verified by sequencing (Qingke, Beijing).
The confirmed recombinant strain is preserved at -80°C in 25% (v/v) glycerol for long-term storage. For routine cultivation, the engineered strain is grown in LB liquid medium containing 100 μg/mL Kana at 37°C with shaking at 150 rpm.
Figure 21: Construction of BL21-TRYP. (A) The plasmid map of pET28a-TRYP; (B) The gene circuit of BL21-TRYP; (C) The flow chart of Construction of BL21-TRYP.
The agarose gel electrophoresis results for the TRYP gene are shown in Figure 22. As indicated by the marker, bands of the correct corresponding lengths were observed for TRYP genes, confirming it's successful expression.
Figure 22: the agarose gel electrophoresis analysis of the TRYP gene fragment
The agarose gel electrophoresis result confirms the
Following the successful construction of the pET-28a-TRYP vector, this experiment aimed to
The TRYP-overexpressing engineered strain was inoculated into 50 mL of LB medium supplemented with
The protein solution was mixed with 5× reducing protein sample buffer at a 4:1 ratio and denatured by boiling for 15 minutes. The proteins were separated on a 12% SDS-PAGE gel at 120 V. Subsequently, the proteins were transferred from the gel to a PVDF membrane under cold conditions.
The membrane was blocked with 5% non-fat milk in TBST for 1 hour at room temperature. To detect the His-tagged protein expressed from the pET vector, the membrane was incubated overnight at 4°C with a mouse anti-His tag primary antibody (1:1000 dilution, Cat AH367, Beyotime). The membrane was then washed three times with TBST for 10 minutes each. Following the washes, it was incubated for 1 hour at room temperature with an HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (1:1000 dilution, Cat A0216, Beyotime).
After another three 10-minute washes with TBST, ECL chemiluminescent substrates A and B were mixed at a 1:1 ratio. The washed PVDF membrane was briefly placed on absorbent paper to remove excess buffer, then transferred to an imaging tray. The prepared ECL solution was applied to completely cover the membrane. After a 1-minute incubation, excess liquid was carefully wicked away, and the membrane was placed into a chemiluminescence imaging system to capture the signal according to the preset program.
Figure 23: This is a flow chart of Western Blot.
Figure 24: SDS-PAGE analysis of recombinant TRYP overexpression in E. coli
The Western Blot result provides
The ultimate goal of our project is to produce functional trypsin. Having successfully expressed the recombinant trypsinogen, the next critical step was to
BL21-pET-28a-TRYP is inoculated into 50 mL LB (100 μg/mL Kana) in 250-mL flasks and cultured at 37°C, 150 rpm.When the OD₆₀₀ of the bacteria reaches 0.6, 0.5 mM IPTG is added and the temperature is shifted to 16°C for 20-hour induction. Cells are harvested by centrifugation (10,000 × g, 5 min), resuspended in ice-cold PBS (pH 7.5), and sonicated (70W, 3-s pulse/1-s interval, 20 min; Jingxin, Shanghai). Inclusion bodies are collected by centrifugation (10,000 × g, 30 min, 4°C).
The pellets are washed with PBS containing 2M urea (A68476, Innochem) and incubated at 25°C, 150 rpm for 1 h. Following centrifugation (10,000 × g, 30 min, 4°C), the pellets are solubilized in denaturation buffer (20 mM Tris, 8 M urea, 20 mM DTT, pH 8.5; 10 mL/g pellet) and incubated at 25°C for 4 h with shaking. Subsequently, the supernatant is collected (12,000 × g, 20 min) and diluted 1:40 in refolding buffer (20 mM Tris, 1 mM cystine, 3 mM cysteine, 1 M urea, pH 9), after which the solution is stirred at 4°C overnight.
Refolded TRYP is concentrated using a 10-kDa ultrafiltration tube (Millipore), resuspended in Tris buffer (40 mM Tris, 0.1 M NaCl, 10 mM CaCl₂, pH 8.0), and activated at 25°C for 1 h. Protein concentration is determined by Bradford assay (P0006, Beyotime). Samples are aliquoted and stored at -80°C.
Figure 25: This is a flow chart of "Trypsin Expression and Refolding".
The experiment is carried out in accordance with the instructions of the Trypsin (TRY) Activity Detection Kit (AKPR005M, boxbio). Trypsin can catalyze the hydrolysis of the ester bond in N-Benzoyl-L-Arginine-Ethylester (BAEE), generating N-Benzoyl-L-Arginine (BA), which has a characteristic absorption peak at 253 nm.
The operation process is as follows:
Intracellular proteins of BL21 (DE3) and BL21-TRYP strains are extracted and added to the reaction system (Oppenheimer et al., 1966). First, preheat a UV spectrophotometer or microplate reader for over 30 minutes, set the wavelength to 253 nm, and calibrate to zero using distilled water. Next, prewarm the detection working solution at 25°C for at least 10 minutes before testing. Then, add reagents sequentially to a 96-well UV plate or micro quartz cuvette: dispense 10 μL of crude enzyme solution into the assay group and 10 μL of distilled water into the blank group, with 200 μL of detection working solution added to both groups. For absorbance measurement: mix thoroughly, start timing immediately, and record the absorbance at 253 nm at 10 seconds (total elapsed time) as A1 (for both assay and blank groups). Incubate at 25°C for exactly 60 seconds, then measure the absorbance at 70 seconds (total elapsed time) as A2 (for both groups). Calculate ΔA for the assay group (ΔAassay = A2assay − A1assay) and the blank group (ΔAblank = A2blank − A1blank), then determine the net ΔA as ΔAassay − ΔAblank. The definition of the enzyme activity unit is: At 25 °C, per mg of protein in a 1 mL reaction system, an increase in the absorbance at 253 nm by 0.0005 per minute is defined as 1 enzyme activity unit (U/mg).
Figure 26: This is a flow chart of "
The absorbance at 253 nm of the intracellular protein of the BL21 (DE3) strain is near zero, and the corresponding enzyme activity is extremely low. the absorbance at 253 nm of the intracellular protein of the BL21-TRYP strain increased significantly, and the enzyme activity reached 1748 U/mg(Figure 27). Compared with the BL21 (DE3) control group, the trypsin activity of the BL21- TRYP strain is greatly improved, and there is an order-of-magnitude difference in absorbance and enzyme activity.
Figure 27 : Comparison of BAEE cleavage activity of intracellular proteins between BL21 (DE3) and TRYP-overexpressing strains
The results provide a
This successful production of a highly active protease from a recombinant source validates our entire workflow, from gene design and expression to protein refolding and activation(Szilagyi et al., 2001). It establishes a solid foundation for the practical application of our enzyme in improving protein digestibility in infant formula.
In summary, we have successfully developed and validated a robust system for producing highly active recombinant trypsin. Our key achievements are:
This complete, validated workflow provides a solid foundation for our project's goal: using this engineered enzyme to pre-hydrolyze casein and improve the digestibility of infant formula(Sampson et al., 1991). It also serves as a valuable case study for future iGEM teams on the production of active proteases in E. coli.
To address the widespread issue of lactose intolerance, this section details our successful engineering of a system for producing functional β-galactosidase (LacZ). Our work followed a systematic, three-step validation process: (1)
To address lactose intolerance, our goal is to produce recombinant β-galactosidase (lacZ). The first step is to
We codon optimized the open reading frame (ORF) of lacZ to meet RFC10 standards, synthesized it and inserted it into the pET-28a plasmid digested by NdeI and XhoI to generate the PET-28A-lacZ recombinant vector. The vector was transformed into E.coli BL21 (DE3) (D1009S, Beyotime, China) strain. Positive clones were screened in LB (Luria Bertani) plates containing 100 μg/mL kanamycin (Kana), and sequencing verification was performed (Beijing, Qingke) to obtain recombinant engineered strains. The strain was stored at -80°C with 25% (v/v) glycerol as the antifreeze. In this experiment, the culture conditions of our engineered strain were 37°C, 150 rpm, and LB liquid medium containing 100 μg/mL kana was used for inoculation and expanded culture.
Figure 28: Construction of BL21-LacZ. (A) The plasmid map of pET28a-LacZ; (B) The gene circuit of BL21-LacZ; (C)The flow chart of Construction of BL21-LacZ.
As shown in Figure 29, there is a distinct band in the lacZ lane at approximately 3072 bp, which corresponds to the expected size of the lacZ gene fragment. This results demonstrate that the lacZ gene was successfully amplified, yielding a specific product of the expected size.
Figure 29: the agarose gel electrophoresis analysis of the lacZ gene fragment
The agarose gel electrophoresis result confirms the
Having successfully constructed the pET-28a-lacZ vector, the next crucial step was to
We analyzed the lacZ expression by using Western blot. We inoculated engineered bacteria overexpressing lacZ into 50 mL LB medium thtat contain
Figure 30: This is a flow chart of Western Blot.
Based on the SDS-PAGE analysis, a distinct protein band was detected in the experimental lane, migrating at the expected molecular weight (116 kDa) corresponding to β-galactosidase(Figure 31). The results verify successful expression of the lacZ-encoded protein, consistent with the expected molecular weight of β-galactosidase. These findings confirm that the recombinant construct enabled efficient production of the target protein.
Figure 31: SDS-PAGE analysis of recombinant lacZ overexpression in E. coli
The Western Blot analysis provides
Having confirmed the successful expression of the lacZ protein, the final and most critical step was to
The purified lacZ enzyme with a final concentration of 0.1 U/mL was added to 1 mL of the lactose reaction system with an initial concentration of 10 mM, incubated at an appropriate temperature, and samples were taken at 0, 2, 4, 6, and 8 hours respectively. The Glucose production was determined by the Abcam Glucose Assay Kit (ab272532) to indirectly reflect lactose consumption. The specific operation is as follows: Add 5 μL of glucose standard or sample to be tested respectively to 1.5 mL centrifuge tubes, then add 500 μL of detection reagent and vortex to mix well. After tightly closing the tube cap, precisely heat in a boiling water bath for 8 minutes, and then cool in an ice water bath for 4 minutes. Take 200 μL of the reaction solution and transfer it to a 96-well plate. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance at a wavelength of 630 nm. Calculate the glucose concentration based on the standard curve and then infer the degradation amount of lactose.
Figure 32: This is a flow chart of measurement of the lactose content lacZ breaks down
The temporal profile of lactose concentration was monitored over an 8-hour period (Figure 33). The initial lactose concentration at 0 h was 10 mM. A gradual decline was observed with time, decreasing to 9.0 ± 0.6 mM at 2 h, followed by a more pronounced reduction to 6.5 ± 0.8 mM at 4 h. By 6 h, the lactose concentration further decreased to 4.0 ± 0.6 mM, and reached its lowest value of 2.0 ± 0.4 mM at 8 h.
Figure 33: Efficacy of recombinant β-galactosidase in lactose degradation.
The results clearly demonstrate that our engineered and purified lacZ protein is
In summary, we have successfully constructed and validated an effective system for producing active β-galactosidase. The key milestones achieved are:
This complete and successful characterization of the existing part