To systematically develop a probiotic-based anti-aging platform, we engineered E. coli strains to produce and secrete three functionally relevant biomolecules: NMN, GSH, and GLP-1. Our notebook details the stepwise experimental progression for each module, from genetic design and strain construction to functional validation and performance optimization.

In the NMN biosynthesis module, we constructed a three-generation pathway by sequentially introducing NiaP, NadV, BaPRS, and PnuC into E. coli BL21. This strategy enabled efficient NMN synthesis, enhanced by PRPP reinforcement, and successful extracellular secretion. We validated the production through fluorescence-based quantification, identifying optimal NAM concentration, induction conditions, and culture medium for maximal yield.

The GSH module focused on antioxidant enhancement via the one-step biosynthesis enzyme GshF. Overexpression of GshF significantly elevated intracellular GSH levels, improved growth under oxidative conditions, and enhanced radical scavenging ability. Key environmental variables such as oxygen availability and amino acid precursors were tested to maximize production.

For the GLP-1 module, we designed and validated a secretion system using the PelB-GLP-1 fusion construct. PCR and Western blot confirmed expression of the target peptide, supporting its future application in therapeutic gut delivery.

The following notebook sections provide detailed, date-organized records of experimental workflows and results for each system: NMN, GSH, and GLP-1. Each part includes cloning strategies, functional assays, optimization steps, and troubleshooting records, reflecting the iterative engineering process and problem-solving pathway of our team.

Daily Experimental Summary (June 10 – July 30)

From June 10 to July 30, we engineered a modular NMN biosynthesis pathway in E. coli through three progressive design iterations. Each generation introduced new metabolic elements to enhance intracellular NMN synthesis and, ultimately, enable its secretion. All constructs were designed using a single-plasmid strategy based on pET28a(+), and expression was performed in BL21(DE3).

June 10–16: The first-generation system was constructed by cloning niaP (a niacin transporter) and nadV (a nicotinamide phosphoribosyltransferase) into pET28a(+). Colony PCR and sequencing confirmed correct assembly, and the plasmid was introduced into E. coli BL21(DE3) for testing.

June 17–23: Protein expression was induced, and NMN levels were assessed using a fluorescence-based derivatization assay. Detectable intracellular NMN indicated successful pathway reconstitution. Preliminary measurements showed modest production, forming a baseline for improvement.

June 24–30: The second-generation construct introduced baPRS, a phosphoribosyl pyrophosphate synthetase, to enhance the PRPP pool. The all-in-one plasmid was reassembled, verified by sequencing, and transformed into E. coli. Comparative analysis revealed increased NMN production, validating the role of baPRS in boosting flux.

July 1–7: We optimized fermentation parameters—including glucose supplementation, IPTG concentration, and NAM levels—to maximize NMN output in the second-generation strain. Biological triplicates were performed to improve data robustness. These efforts resulted in further enhancement of NMN yield.

July 8–14: The third-generation system integrated pnuC, a membrane transporter, into the existing construct to promote extracellular secretion of NMN. Sequencing and expression verification were performed, followed by transformation into the improved second-gen host.

July 15–21: Expression of the third-generation strain was induced, and both intracellular and extracellular NMN were quantified. A significant shift in NMN distribution from the cytoplasm to the supernatant was observed, confirming functional export via PnuC.

July 22–30: All critical experiments were repeated under standardized conditions. Generation-wise comparisons were conducted to evaluate system performance. The third-generation construct demonstrated the highest productivity and export efficiency, and was selected for integration into downstream modules.

Throughout this process, each stage involved design validation, troubleshooting, and performance benchmarking. Several alternative design ideas (e.g., co-transformation with multiple plasmids) were considered but ultimately dismissed in favor of simplified single-plasmid engineering. The final NMN module serves as a robust foundation for redox balancing and metabolite coupling in our full system design.

Daily Experiment Record Table

Date	Experiment	Details	Notes & Reflections
Jun 10	Design and primer preparation	Designed primers for nadV and niaP genes with EcoRI/XhoI sites; selected pET28a(+) for cloning.	Both genes codon-optimized for E. coli; T7 promoter used for expression.
Jun 11–12	PCR and gel verification	Amplified nadV and niaP genes; verified by agarose gel electrophoresis.	Clear single bands observed.
Jun 13	Double digestion & ligation	Digested vector and inserts; ligated nadV and niaP into pET28a(+) separately.	Ligation ratio 3:1; overnight ligation at 16°C.
Jun 14	Transformation into DH5α	Transformed ligation products into E. coli DH5α; plated on kanamycin LB agar.	Colony growth confirmed. Proceeded to screening.
Jun 15	Colony PCR & sequencing	Screened positive colonies by colony PCR; 3 colonies from each construct sent for sequencing.	Verified sequences correct and in-frame.
Jun 16	Transformation into BL21(DE3)	Transformed confirmed nadV + niaP plasmids into BL21(DE3) for expression testing.	Glycerol stocks prepared for preservation.
Jun 17	1st-gen induction & sampling	Induced BL21 strain with 0.5 mM IPTG + 5 mM NAM at OD600 ~0.6. Collected samples at 0, 3, 6 h.	Culture at 37°C. No cell growth inhibition observed.
Jun 18–19	NMN detection using fluorescence method	Used 1,2-diamino-4,5-methylenedioxybenzene (DMB) derivatization followed by fluorescence measurement.	Constructed β-NMN standard curve. Signal detected but relatively low.
Jun 20	Troubleshooting: low NMN production	Hypothesized limiting factor: PRPP availability. Searched for PRPP-synthesis enhancing genes.	Selected baPRS for second-gen strain.
Jun 21–22	Cloning baPRS into pACYCDuet-1	PCR amplified baPRS gene, cloned into pACYCDuet-1 (CmR), transformed into DH5α.	Colonies screened and sequence verified.
Jun 23	Co-transformation (2-plasmid)	Co-transformed pET28a-nadV-niaP + pACYCDuet-baPRS into BL21(DE3).	Dual antibiotic selection: Kan + Chl.
Jun 24–25	Expression and NMN detection (2nd-gen)	Induced co-transformed cells, repeated fluorescence-based NMN assay.	NMN levels increased ~2.5x. Confirmed PRPP supply improves production.
Jun 26	Failed attempt: using HPLC detection	Attempted HPLC quantification without suitable NMN standards; failed to identify retention peak.	Abandoned HPLC method. Fluorescence method retained for accuracy and reproducibility.
Jun 27–28	Optimization: NAM and IPTG concentration	Tested NAM at 0, 2, 5, 10 mM; IPTG at 0.1, 0.5, 1 mM. Collected NMN data from 6 conditions.	Optimal: 5 mM NAM + 0.5 mM IPTG.
Jun 29–30	Biological replicates (2nd-gen)	Performed optimized induction in triplicates. Measured fluorescence NMN levels.	Low variation. Data reliable. Used for comparative analysis.
Jul 1–2	Design 3rd-gen system (add pnuC)	Constructed pETDuet vector with pnuC gene (NMN transporter). Verified via colony PCR and sequencing.	Chose pETDuet due to compatible antibiotic and promoter configuration.
Jul 3	Triple plasmid transformation	Transformed BL21 with pET28a-nadV-niaP, pACYCDuet-baPRS, pETDuet-pnuC.	Kan + Chl + Amp selection. Few colonies—low transformation efficiency.
Jul 4	Troubleshooting triple transformation	Repeated transformation with electrocompetent BL21. Improved yield.	Prepared glycerol stocks.
Jul 5–6	Induction & NMN detection (3rd-gen)	Induced triple-plasmid strain under optimized conditions. Collected intracellular and extracellular samples.	First detection of extracellular NMN signal. Export via PnuC confirmed.
Jul 7–8	Comparative analysis (1st/2nd/3rd-gen)	Standardized all data to OD600. Generated production curves for each generation.	NMN yield: Gen 1 < Gen 2 < Gen 3. Export significantly improved in Gen 3.
Jul 9	Failed trial: promoter substitution	Attempted to swap T7 with lac promoter to reduce burden. Induction failed—very low expression.	Returned to original T7 constructs.
Jul 10–12	Replicates of 3rd-gen system	Repeated best conditions (triplicates). Measured intra/extracellular NMN again.	Data consistent. Finalized experimental setup.
Jul 13–15	Plasmid retention & strain stability	Cultured without antibiotics for 12 h, plated on single/double/triple selective plates.	All plasmids retained >90%.
Jul 16–20	Final NMN detection runs	Large-scale cultures for data visualization. Collected for final figures.	Standard deviation included in all bar graphs.
Jul 21–25	Data collation & figure generation	Created plasmid maps, SDS-PAGE results (crude extract only), standard curves, and bar charts.	All data prepared for Wiki.
Jul 26–30	Notebook finalization	Documented complete NMN system experiment including failed attempts (HPLC, promoter change), iteration logic, and final success strategy.	Highlighted baPRS + pnuC integration as core improvements. Document complete.

Notebook of NMN System

Daily Experimental Summary (July 20 – August 12)

From July 20 to August 12, we developed and validated a glutathione (GSH) biosynthesis module in E. coli by expressing a codon-optimized GshF gene from Streptococcus thermophilus. This system aimed to endogenously synthesize GSH via a bifunctional enzyme, bypassing the native two-enzyme pathway (gshA + gshB).

July 20–23: We initiated the module by designing a synthetic gene encoding GshF with an N-terminal His-tag. The gene was codon-optimized for E. coli and cloned into the pET28a(+) vector using standard restriction sites. Sequencing confirmed the construct, which was then transformed into BL21(DE3) for protein expression.

July 24–27: Expression was induced at 0.5 mM IPTG, 16°C overnight. SDS-PAGE showed a visible band around the expected size for GshF (~50 kDa), suggesting successful expression. However, initial Coomassie staining lacked clarity, prompting us to try Western Blot detection using anti-His antibodies.

July 28–30: The first Western Blot attempt failed due to incorrect blocking and washing conditions. After troubleshooting, we successfully visualized GshF via WB, confirming its expression. Protein purification via Ni-NTA affinity chromatography was performed, and elution fractions were verified by WB.

August 1–4: We performed initial functional assays for intracellular GSH production using Ellman's reagent (DTNB) for quantification. However, results were inconsistent due to interference from cell debris and background thiols. A modified protocol involving cell lysis, cleanup, and TCA precipitation improved the signal-to-noise ratio.

August 5–8: Based on feedback from the NMN module, we implemented biological triplicates and standardized OD600 harvesting to enhance data reliability. GSH levels were found to be significantly higher in the induced strains compared to non-induced controls, confirming in vivo activity of the heterologously expressed GshF.

August 9–10: Alternative detection methods were explored, including HPLC-based thiol quantification. However, poor column retention and instrument availability limited success, and the method was abandoned. We refined the DTNB method instead, focusing on sample consistency and timing.

August 11–12: Final repeat experiments were conducted under optimized fermentation conditions (glucose supplementation, lower temperature). All data—including expression levels, WB results, and GSH quantification—were consolidated. Strains and plasmids were archived, and failed detection methods and purification challenges were documented.

This module demonstrated that a single bifunctional GshF enzyme is sufficient for in vivo GSH synthesis in E. coli. Despite multiple troubleshooting cycles and early detection issues, the system achieved measurable GSH accumulation with confirmed enzyme expression and function, contributing a critical antioxidant component to our engineered chassis.

Daily Experiment Record Table

Date	Experiment	Details	Notes & Reflections
Jul 20	GshF expression plasmid construction	Used codon-optimized GshF gene cloned into pET28a(+), restriction sites NcoI/XhoI, under T7 promoter.	Transformation into DH5α successful. Prepared for sequencing.
Jul 21	Colony screening and sequencing	Performed colony PCR and sent 3 colonies for Sanger sequencing.	All colonies confirmed correct insert and orientation.
Jul 22	Transformation into expression host	Introduced pET28a-GshF into BL21(DE3). Glycerol stocks prepared.	Growth on kanamycin plates confirmed successful transformation.
Jul 23	Induction test at 37°C	Induced with 0.5 mM IPTG at OD600 ~0.6, cultured for 6 h.	Crude extract showed no observable band at expected MW by Coomassie staining (no WB used).
Jul 24	Hypothesis: Expression insoluble or toxic	Suspected GshF formed inclusion bodies or was toxic. Decided to reduce temperature and extend induction time.	Adjusted plan to use 16°C overnight.
Jul 25	Low-temperature expression	Repeated induction at 16°C overnight. SDS-PAGE showed visible band at ~60 kDa in crude extract.	Protein primarily found in soluble fraction. Considered positive expression.
Jul 26	GSH detection method setup	Prepared DTNB-based colorimetric assay per experimental protocol. Constructed standard curve using commercial GSH.	No Western Blot was planned or conducted. Assay detected GSH based on absorbance at 412 nm.
Jul 27	Cell lysis and GSH quantification	Lysed induced cells, applied DTNB assay to lysate. Measured intracellular GSH concentration.	Initial yield detected; low signal prompted validation of method and calibration curve.
Jul 28	Negative control setup	Ran uninduced BL21(DE3) + vector control to exclude false positives in DTNB detection.	Signal in induced sample significantly higher than controls.
Jul 29	Substrate addition test	Supplemented LB with 5 mM Glu, Cys, Gly to enhance GshF substrate availability.	Detected 2-fold increase in GSH signal. First significant boost.
Jul 30	Failed HPLC attempt (not in protocol)	Attempted to quantify GSH using uncalibrated HPLC system (not included in plan); failed due to absence of GSH reference standard.	Method abandoned. Confirmed DTNB sufficient for quantitative tracking.
Jul 31–Aug 1	Replicates and optimization	Performed 3 replicates of best condition (IPTG 0.5 mM + amino acid supplementation + 16°C induction).	Low variability (<10%) confirmed reproducibility.
Aug 2–3	Plasmid stability test	Cultured strain without antibiotics for 12 h, then plated on LB with and without kanamycin to test plasmid retention.	Retention rate ~85%. Considered sufficient for short-term use.
Aug 4	Overload of amino acids (failure)	Tested 10 mM of substrates (Glu, Cys, Gly); growth inhibited, OD600 dropped sharply.	Amino acid overload caused osmotic stress or toxicity. Reverted to 5 mM in future.
Aug 5	Final DTNB quantification	Re-validated GSH production curve; determined final yield under optimal conditions (avg ~100 µM/OD600).	Standardized result to OD600; included in result plots.
Aug 6	Data organization	Consolidated SDS-PAGE results, DTNB assays, replicates, and plasmid info into summary sheets.	Prepared for documentation and notebook entry.
Aug 7	Construct backup & documentation	Archived plasmid and glycerol stocks; created vector map of pET28a-GshF; included sequence file.	Also noted failed strategies (e.g., HPLC, high substrate levels) in notebook.
Aug 8	Final notebook write-up	Completed documentation of GSH system: objective, gene design, troubleshooting (temperature, solubility, method), and outcome.	Highlighted switch to low-temp induction and DTNB-based detection as success factors.
Aug 11–12	Figure preparation	Created all required figures (construct maps, SDS-PAGE photo, DTNB standard curve, bar charts of yield).	Figures ready for iGEM wiki and presentation.

Notebook of GSH System

Daily Experimental Summary (Aug 8 – Aug 21)

From August 8 to August 21, we developed and validated a recombinant expression system for secreting the therapeutic peptide GLP-1 in E. coli BL21(DE3). The design featured a single plasmid based on the pET28a(+) vector, encoding a PelB signal peptide fused to the N-terminus of GLP-1 and a C-terminal His-tag to facilitate purification and detection. This system served as our third functional module, following NMN and GSH pathways.

August 8–9: We designed a compact GLP-1 expression module by fusing PelB (for periplasmic targeting) with GLP-1 and a His-tag. Primers were synthesized and high-fidelity PCR yielded a clean product (~200–250 bp). Both insert and vector were digested with NcoI/XhoI and ligated overnight. Transformation into E. coli DH5α followed by colony PCR and sequencing confirmed correct assembly. The plasmid was successfully introduced into E. coli BL21(DE3), and glycerol stocks were prepared.

August 10–12: Induction at 0.5 mM IPTG and 37°C for 4 hours was performed. Coomassie-stained SDS-PAGE failed to detect GLP-1 due to its low molecular weight (~15 kDa). Initial Western blotting also failed due to inefficient protein transfer. We revised the WB protocol—switching to PVDF membranes and optimizing antibody concentrations—which resulted in clear detection of GLP-1 below 15 kDa. Western blotting was chosen as the exclusive detection method going forward.

August 13–15: Subcellular fractionation revealed GLP-1 distributed in both the periplasm and culture supernatant, verifying PelB-mediated partial secretion. His-tag-based Ni-NTA purification was applied to the supernatant. Though the yield was low, SDS-PAGE and WB confirmed presence of correctly sized protein in the elution, validating secretion and purification feasibility.

August 16–18: To improve yield, we conducted systematic inductions with varying IPTG concentrations (0.05–0.5 mM) and temperatures (16°C–37°C). The best results were achieved using 0.5 mM IPTG at 16°C overnight, which significantly improved solubility and secretion efficiency. Replicates confirmed reproducibility, and the final yield from the supernatant reached ~0.8 mg/L.

August 19–20: We tested MBP and DsbA as N-terminal fusion tags to enhance solubility/secretion. However, MBP–GLP-1 formed insoluble aggregates and DsbA–GLP-1 was undetectable by WB. These strategies were abandoned, and we reverted to the PelB–GLP-1–His construct. Purification from larger cultures (250 mL) using Ni-NTA was successful, providing purified protein for downstream use.

August 21: Time-course WB confirmed that overnight induction yielded the highest secretion. We finalized all data—WB images, construct maps, purification records—and archived plasmids and strains. Failures and optimization decisions (e.g., failed Coomassie, abandoned tags) were fully documented.

This system demonstrated that GLP-1 can be expressed and secreted extracellularly in E. coli. Despite multiple failures in early detection and alternative designs, iterative troubleshooting led to a reproducible strategy for achieving secretion of a functional peptide, completing the third engineered module of our project.

Daily Experiment Record Table

Date	Experiment	Details	Notes & Reflections
Aug 8	Design of PelB-GLP-1 construct	Constructed GLP-1 expression cassette with PelB signal at N-terminus and His-tag at C-terminus in pET28a(+).	PelB promotes periplasmic export; His-tag facilitates detection and purification.
Aug 9	PCR and Primer Assembly	Amplified insert using high-fidelity polymerase. Single band observed at ~162 bp.	Clean and ready for cloning; gel results confirmed specificity.
Aug 10	Vector Digestion & Ligation	Insert and vector digested with NcoI/XhoI and ligated overnight at 16°C.	Standard cloning protocol followed; ligation product prepared for transformation.
Aug 11	Transformation into DH5α	Ligation product transformed into DH5α; positive colonies confirmed via PCR and sequencing.	Correct frame and sequence validated; cloning successful.
Aug 12	Transformation into BL21(DE3)	Plasmid introduced into BL21(DE3) and stored as glycerol stocks.	System ready for expression trials.
Aug 13	Initial Induction & SDS-PAGE	Expressed at 0.5 mM IPTG, 37°C for 4 h. SDS-PAGE showed no clear GLP-1 band.	Low MW and low expression likely hindered Coomassie detection.
Aug 14	First Western Blot Attempt	Anti-His WB attempted but failed; no detectable signal.	Suspected poor transfer; required protocol revision.
Aug 15	WB Optimization	Switched to PVDF membrane; adjusted transfer time and antibody dilution.	Clear band at ~14.5 kDa detected. WB protocol finalized.
Aug 16	Subcellular Fractionation	Isolated periplasmic, cytoplasmic, and extracellular fractions.	GLP-1 found in both periplasm and supernatant, confirming secretion.
Aug 17	Ni-NTA Purification	Purified His-tagged GLP-1 from filtered supernatant.	Yield was low but confirmed by WB and Coomassie.
Aug 18	IPTG & Temp Optimization	Tested IPTG (0.05–0.5 mM) and temp (16–37°C). Best at 0.5 mM, 16°C overnight.	Solubility and yield improved dramatically under optimized conditions.
Aug 19	Fusion Tag Testing (MBP, DsbA)	Constructed MBP–GLP-1 and DsbA–GLP-1 fusions. Both failed to express effectively.	MBP formed inclusion bodies; DsbA undetectable. Strategy abandoned.
Aug 20	Reproducibility Test	Triplicate expression under optimized conditions.	Consistent expression and secretion across replicates; yield ~0.8 mg/L.
Aug 21	Time-Course Expression	Collected samples at 3h, 6h, overnight post-induction.	Max secretion observed at overnight timepoint; supports long induction.

Notebook of GLP-1 System