iGEM 2024 — Team nefu-china

![](https://static.igem.wiki/teams/5313/home/homegotop.png)

[Electroporation for Knockout of the *guab* Gene in *E. coli* BW25113](#side1)
[Mutagenesis of *ndmB*](#side2)
[Construction, Validation, and Screening of Sensor](#side3)
[Production of 7-MX](#side4)
[Coffee Grounds](#side5)

Notebook

Electroporation for Knockout of the *guab* Gene in *E. coli* BW25113

5.1-5.8 — Electroporation to Knockout *guab* Gene in *E. coli* BW25113

* Preparation of BW/pKD46 Electrocompetent Cells:
* Introduced the up-kan-down targeting fragment into the prepared competent cells.
* Performed colony PCR verification; the expected target band was 600 bp, but the results were incorrect.

5.9-5.15 — Gene Knockout

Replacement of Primers and Colony PCR:

* New primers were designed and used to perform another round of colony PCR to improve the accuracy of the *guab* gene knockout verification.

Selection of Single Clones for Validation:

* Three single colonies (No. 6, No. 10, and No. 14) were chosen for further validation of the *guab* gene knockout.

Validation on Selective Media:

* The selected clones were cultured on M9 minimal medium plates, with or without the addition of 0.2 mM xanthine, to assess their growth.

Incubation:

* The cultures were incubated in a shaking incubator at 37°C for 24 h to monitor the growth patterns and confirm the gene knockout.

The growth results on the selective media will help us determine if the *guab* gene has been successfully knocked out in the selected clones.

![](https://static.igem.wiki/teams/5313/notebook/notebook1.png)

5.16-5.22 — Gene Knockout Verification

Verification by Streaking Method:

* The selected E. coli BW25113 clones were streaked onto agar plates to validate the knockout of the *guab* gene.

Observation of Growth Results:

* The results confirmed successful gene knockout. The clones were able to survive on the medium containing xanthine, but showed no growth on the medium without xanthine.

These data indicated that the *guab* gene knockout was successful, as the knockout strains exhibited the expected growth dependency on xanthine

![](https://static.igem.wiki/teams/5313/notebook/notebook2.png)

5.23-5.29 — Gene Knockout

Streaking on Kanamycin-Resistant Medium:

* The verified *guab* knockout strains were streaked onto agar plates containing kanamycin to assess their resistance and confirm the presence of the knockout cassette.

Selection and Spotting of Single Clones:

* Single colonies were picked and first inoculated on a plate with A+K (ampicillin and kanamycin) resistant medium, followed by inoculation on a plate with K (kanamycin only) resistant mediumto evaluate their growth patterns.

Identification of Correct Knockout Strains:

* Strains that could grow on kanamycin (K) plates but failed to grow on A+K plates were selected for further propagation. These growth patterns indicate the successful knockout of the *guab* gene and the appropriate loss of the pKD46 plasmid (ampicillin resistance).

![](https://static.igem.wiki/teams/5313/notebook/notebook3.png)

This procedure helps us confirmthat the strains are correctly knocked out for the *guab* gene and do not carry any unwanted plasmid, which is critical for subsequent experimental steps.

5.30-6.5 — Gene Knockout

Preparation of Competent Cells:

* The verified *guab* knockout strain (BW25113 *Δguab*) was prepared as competent cells for further transformation.

Transformation with pYB1s-ndmDBACE Plasmid:

* The plasmid pYB1s-ndmDBACE was introduced into the competent BW25113 *Δguab* strain.
* After transformation, the cells were incubated in a 37°C shakerfor 16 h. However, the transformed strains could poorly grow.

Revival and Re-plating:

* The BW25113 *Δguab* strain was revived and spread onto K30+S (kanamycin and spectinomycin) resistant agar plates to promote better growth conditions.

Re-transformation with pYB1s-ndmDCBAE Plasmid:

* The pYB1s-ndmDCBAE plasmid was reintroduced into the BW25113 *Δguab* strain to ensure successful transformation.

These steps aim to optimize the transformation conditions and confirm the stable integration of the desired plasmid into the *guab* knockout strain for subsequent experiments.

6.6-6.13 — Gene Knockout

Comparison of Growth Curves:

* Conducted a comparative analysis of the growth curves between the wild-type E. coli BW25113 strain and the *guab* knockout strain (BW25113 *Δguab*).

Observation and Analysis:

* The growth patterns were monitored under identical conditions to evaluate the impact of the *guab* gene knockout on bacterial growth.
* Differences in growth rates, lag phases, and overall cell density were carefully recorded to assess the physiological effects of the gene knockout.

![](https://static.igem.wiki/teams/5313/notebook/notebook4.png)

This comparison provides crucial insights into the metabolic and growth characteristics of the *guab* knockout strain, confirming the gene knockout's effects and helping to guide further optimization in the engineered strain's use for downstream applications.

Mutagenesis of *ndmB*

5.16-5.22 — Exploration of Random Mutagenesis Conditions for *ndmB*

Targeted Site-Directed Mutagenesis:

* Specific pocket residues within the NdmB enzyme were selected for site-directed mutagenesis to potentially enhance the enzyme's catalytic activity.
* Mutations were introduced at these sites to explore their impact on the enzyme's structure and function.

Detection Using Enzyme Assays:

* The activity of the mutated NdmB variants was assessed using a plate reader.
* This initial screening aimed to identify any changes in enzymatic activity due to the introduced mutations.

HPLC Analysis:

* High-Performance Liquid Chromatography (HPLC) was employed to detect the production of 7-MX or other relevant metabolites.
* Unfortunately, the HPLC analysis showed no detectable product formation, suggesting that the mutations did not enhance the enzyme's activity.

Optimization of PCR Conditions:

* To improve the mutagenesis efficiency, PCR conditions were adjusted, specifically modifying the concentrations of Mn2+ and dNTPs:

![](https://static.igem.wiki/teams/5313/notebook/notebook5.png)

0.01 mM Mn2+ with dNTPs

![](https://static.igem.wiki/teams/5313/notebook/notebook6.png)

0.02 mM Mn2+ without dNTPs

![](https://static.igem.wiki/teams/5313/notebook/notebook7.png)

0.01 mM Mn2+ without dNTPs

![](https://static.igem.wiki/teams/5313/notebook/notebook8.png)

These steps aimed to generate NdmB mutants with enhanced functionality, but further optimization and additional mutagenesis strategies may be required to achieve the desired increase in enzyme activity and product yield.

* Use gel extraction to recover the vector and mutated fragments.
* Perform Gibson assembly (C116).
* Transform the competent cells, extract plasmids, send plasmids for DNA sequencing, and check the mutation rate.

![](https://static.igem.wiki/teams/5313/notebook/notebook9.png)

Based on the results, we found that mutations only occurred in the SPRBCC region under the condition of 0.02 mM Mn2+.

Therefore, subsequent experiments will continue to use 0.02 mM Mn2+.

5.23-5.29 — Verification of Random Mutagenesis Conditions for *ndmB*

PCR with 0.02 mM Mn2+ and dNTPs:

![](https://static.igem.wiki/teams/5313/notebook/notebook10.png)

PCR with 0.02 mM Mn2+ without dNTPs:

![](https://static.igem.wiki/teams/5313/notebook/notebook11.png)

* Gel extraction was used to recover the vector and mutated fragments.
* Gibson assembly (C116) was performed.
* Transformed cells were cultured, plasmids extracted, sent for sequencing, and mutation rates were checked.

There were many cloned with unmutated sequences.

5.30-6.5 — Verification of *ndmB* Random Mutagenesis Conditions 2

Site-Directed Mutagenesis of *ndmB*:

* Site-directed mutagenesis was performed at potential pocket sites on NdmB (2012).
* Degenerate primers were used for PCR on the plasmid (pYB1s-ndmDtB) to induce mutations at Trp256, Cys267, and Met27.
* The product was purified using DpnI digestion and transformed into BW25113 for functional validation.

![](https://static.igem.wiki/teams/5313/notebook/notebook12.png)

Verification of Random Mutagenesis Conditions for *ndmB*:

* Gel extraction was used to recover the vector and mutated fragments.
* Gibson assembly (C116) was performed.
* Single colonies were picked, plasmids were extracted followed by DNA sequencing, and mutation rates were evaluated.

![](https://static.igem.wiki/teams/5313/notebook/notebook133.png)

Construction, Validation, and Screening of Sensor

5.30-6.5 — Construction of pYB1s-ndmDCE Plasmid

Construction of pYB1s-ndmDCE Plasmid:

* The pYB1s-ndmDCE plasmid was constructed and transformed into BW25113-*△guaB*.
* If caffeine degradation is efficient, the produced PX will be converted into xanthine, allowing E. coli to survive.
* If caffeine degradation is inefficient, a large amount of caffeine will be converted into 3-MX, which does not support E. coli surviving.

Colony PCR results (using ndmA-F and ndmA-R primers):

![](https://static.igem.wiki/teams/5313/notebook/notebook14.png)

Functional Validation of pYB1s-ndmDCE Plasmid:

* The mutated plasmid pYB1s-ndmDtBs was obtained.
* Expression was induced in deep-well plates.
* Supernatant from the transformed bacteria was transferred to a 96-well plate, and BW-*△guaB*+DCE was added.
* Growth curves were generated .

6.6-6.13 — Functional Validation of pYB1s-ndmDCE

* The correctly sequenced pYB1s-ndmDCE plasmid was transformed into BW*△guaB*, and single colonies were picked for induction.
* ZY medium containing xanthine was removed and the bacteria were washed; the supernatant was discarded after centrifugation, and cells were washed once with M9 medium. The cells were then resuspended in 5 mL of M9 medium.

6.14-6.19 — Feasibility Testing of Current Conditions for Screening NdmBs

* Verified whether induction at 25°C in deep-well plates (ZYM-5052) and whole-cell catalysis in M9 medium were feasible.
* pYB1s-ndmDtA plasmid was transformed into BW25113 as a high-efficiency control group.
* pYB1s-ndmDtB plasmid was transformed into BW25113 as an experimental group.
* pYB1s-ndmDtA showed nearly 100% conversion of TP to 3-MX, proving the feasibility of the method.
* pYB1s-ndmDtB showed an average conversion rate of 6.9% from TP to 1-MX.

![](https://static.igem.wiki/teams/5313/notebook/notebook145.png)

Comparison of pYB1s-ndmDtA and pYB1s-ndmDtB.

![](https://static.igem.wiki/teams/5313/notebook/notebook15.png)

* Induced pYB1s-ndmDCE (BW25113) by adding a small amount of xanthine powder.
* After induction, washed cells with M9 medium and inoculated 10% of the bacteria into M9 medium containing 7-MX.
* Incubation for 36 h to observe difference between media with and without 7-MX.
* Growth inhibition or cell death only began after initial growth (possibly due to incomplete dissolution of 7-MX during induction); without 7-MX, growth was very slow.
* LB medium's yeast extract also contains some intermediate metabolites like xanthine; different amounts of yeast extract were added, and growth curves were plotted in a 96-well plate.

![](https://static.igem.wiki/teams/5313/notebook/notebook16.png)

* The results showed that the xanthine content in the LB medium was insufficient, and doubling the yeast extract could better supplement it.
* Subsequently, the “LB Y2” medium (with double the yeast extract of LB) was used to cultivate and induce the strains.

Validation of 7-MX specificity of biosensors.

* **pYB1s-ndmDtBA** can degrade caffeine into 7-MX.
* **pYB1s-ndmDCE** can further degrade 7-MX into xanthine.
* Used **pYB1s-NdmDtBA** as the production strain and **pYB1s-ndmDCE** as the testing strain.

Testing of pYB1s-ndmDCE:

* Introduced different substrates (compounds in the xanthine family ) to the BW25113 strain with the *guab* gene knocked out, and examine bacterial growth status.
* The growth status of the strain was poor, indicating that this testing method was not effective to be used for the specificity verification of 7-MX.

![](https://static.igem.wiki/teams/5313/notebook/notebook17.png)

6.20-6.26 — Caffeine to 7-MX Production

* **pYB1s-ndmDtBA** can degrade caffeine into 7-MX.
* **pYB1s-ndmDCE** can degrade 7-MX into xanthine.
* Used **pYB1s-ndmDtBA** as the production strain and **pYB1s-ndmDCE** as the testing strain.
* The first round of testing was completed.
* The first round of testing showed poor growth status of the strain when measuring OD values using a microplate reader.

7.11-7.17 — Caffeine Conversion to 7-MX

* Analyzed the second round of screening results and identified an advantageous strain, 4-C5.
![](https://static.igem.wiki/teams/5313/notebook/notebook18.png)
![](https://static.igem.wiki/teams/5313/notebook/notebook19.png)* Conducted sequence comparisons between the two rounds of results, identifying position 289 as the most crucial residue .

7.18-7.24 — Caffeine Production of Hypoxanthine

Used **pYB1s-ndmDtBA** as the production strain and **pYB1s-ndmDCE** as the test strain, conducting three rounds of screening (only targeting mutations at position 289).

* The mutated plasmid was first transformed into DH5α, with single colonies grown and washed in water; plasmids were extracted and then transformed into BW25113, spread on square plates.
* Single colonies were picked and inoculated into deep-well plates (800 μL of ZY + arabinose + IPTG + S50), with the first four wells containing non-mutated strains, and then induced the bacteria at 25°C for 18 h.
* Whole-cell catalysis was conducted at 25°C for 18 h.
* After centrifugation, 200 μL of supernatant was added to a 96-well plate with 10 μL of a supplement solution (containing glucose, calcium chloride, magnesium chloride, trace elements, inducers, and antibiotics).
* An additional 10 μL of the induced DCE was added.

  (Due to the apparent slow growth of DCEA, delayed induction may be required; the 96-well plate was sealed in a plastic bag and stored at 4°C until DCE induction was complete.)
* Growth was measured using a microplate reader, with successful growth indicating the ability to produce 7-MX.

![](https://static.igem.wiki/teams/5313/notebook/notebook200.png)

Analysis of the second-round screening results:

* Selected some samples for sequencing.
* The relationship between amino acids and yield after mutation was analyzed by GPT.

![](https://static.igem.wiki/teams/5313/notebook/notebook211.png)

Production of 7-MX

8.1-8.5 — Exploration of Better Expression Conditions

* Tested different Anderson strong promoters for ***ndmBs***.
* After construction, liquid-phase testing revealed the presence of by-products during 7-MX production.

![](https://static.igem.wiki/teams/5313/notebook/notebook222.png)

Screening of NdmBs Mutants:

* After plasmid transformation into strains, single colonies were separated by streaking on plates, and the plasmid was extracted and digested with KpnI and XbaI. The correct bands were identified at 2448 and 3272, which were verified by DNA sequencing analysis.

![](https://static.igem.wiki/teams/5313/notebook/notebook23.png)

1. pYB1s-ndmDtBs X289 was transformed into BW25113.

2. pYB1s-ndmDtBs X289 with CRS and pSB1c-frmAB-FDH were co-transformed into BW25113.

Induction at 25°C for 18 h:

Single transformation results:

1-G2 (CCC-289): Pro=P289 - No bacterial growth.

2-G2 (TCA-289): Ser=S289 - Cell lysis upon induction.

1-B12 (GCA-289): Ala=1A289 - OD=13.7, white color, foul odor, apparently contaminated .

2-B12 (GCA-289): Ala=2A289 - Normal OD=7.45.

Protein analysis planned.

8.6-8.8 — Mutant Testing

* Induction and whole-cell catalysis were performed on 1-G2, 2-G2, 1-B12, and 2-B12 strains, and samples were prepared for HPLC.
* SDS-PAGE was conducted.

![](https://static.igem.wiki/teams/5313/notebook/notebook244.png)

8.8-8.12 — Whole-Cell Catalysis of Mutants

* Repeated whole-cell catalysis for 1-G2 and 2-G2 strains (unfortunately contaminated).

8.19-8.22 — Whole-Cell Catalysis of B12 (Q289Ala) *ndmBs* Strain

* Whole-cell catalysis (20°C, 20 h): Fermentation broth was freshly prepared (caffeine + Tris-HCl pH 9) and operated on ice.
* OD gradient (8 mM): 30, 50, 70, 100.
* Substrate gradient (50 OD): 1, 2, 4, 8, 10.
* Samples were prepared for testing under the best conditions.

1. Induction of B12 *ndmBs* + CRS dual-transformation strain is in progress.

2. Transformation and plating of the new C12 (289Ala) strain is underway.

8.22-8.26 — C12 and B12 *ndmBs* Single Transformation Strains, C12 *ndmBs*+CRS Dual Transformation Strains Running HPLC

* Induction: Cultivated at 37°C for 1.5 h before adding the inducer until OD was between 0.2 and 0.6.
* Transferred to 25°C, induced for a total of 18 h, and the final OD was approximately 7-8.
* Whole-Cell Catalysis: Whole-cell catalysis (20°C, 20 h) involved transferring the cell suspension (10 mL centrifuge tube with 1 mL fermentation broth) to a 100 mL Erlenmeyer flask and shaking.
* OD gradient (10 mM): 10, 20, 40, 80, 100.
* Substrate gradient (50 OD): 1, 2, 4, 8, 10.
* Sample Preparation: Directly added Tris-HCl (pH 9) to the whole-cell catalysis completion flasks, diluted to a substrate concentration of 2 mM, and added 4 mL Tris-HCl to the 10 mM substrate group, reaching a total volume of 5 mL. Similarly, diluted the 4 mM substrate to a total volume of 2 mL.
* Samples were mixed, 1 mL was transferred to an Eppendorf tube, centrifuged at 10,000 g for 10 min, and the supernatant was passed through a 0.22 μm filter membrane into HPLC vials.
* The yieldsand time curve were obtained.

![](https://static.igem.wiki/teams/5313/notebook/notebook25.png)

1. Induction of B12 *ndmBs* + CRS dual-transformation strain is in progress.

8.27-9.1 — Second Round of Fermentation Condition Optimization

* Focused on optimizing the production of the B12 strain based on the first round of fermentation conditions.
* Re-induced and fermented:

Induction: Cultivated at 37°C for 1.5 h before adding the inducer until OD was between 0.2 and 0.6.
Transferred to 25°C, starting from the time of transferring at 37°C, induced for a total of 18 h, and the final OD was approximately 7-8.

* Whole-Cell Catalysis: Whole-cell catalysis (20°C, 20 h) involved transferring the cell suspension (10 mL centrifuge tube with 1 mL fermentation broth) to a 100 mL Erlenmeyer flask and shaking.
* OD gradient (10 mM): 10, 20, 40, 80, 100.
* Substrate gradient (50 OD): 1, 2, 4, 8, 10.

![](https://static.igem.wiki/teams/5313/notebook/notebook26.png)
![](https://static.igem.wiki/teams/5313/notebook/notebook27.png)

* Sample Preparation: Directly added Tris-HCl (pH 9) to the whole-cell catalysis completion flasks, diluted to a substrate concentration of 2 mM, and added 4 mL Tris-HCl to the 10 mM substrate group, reaching a total volume of 5 mL. Similarly, diluted the 4 mM substrate to a total volume of 2 mL.
* Samples were mixed, 1 mL was transferred to an Eppendorf tube, centrifuged at 10,000 g for 10 minutes, and the supernatant was passed through a 0.22 μm filter membrane into HPLC vials.

Coffee Grounds

8.19-8.22 — Extraction of Caffeine from Dry Coffee Grounds

* Collected coffee grounds from various stores, including Starbucks, Luckin, Kudi, 818, KFC, and Shengji.

![](https://static.igem.wiki/teams/5313/notebook/notebook28.png)
![](https://static.igem.wiki/teams/5313/notebook/notebook29.png)

* Dried the grounds for a day but found that they were still moist.
* Measured 10 g of coffee grounds from each brand, placed them in Erlenmeyer flasks, and dried them.
* Added water at four times the mass-to-volume ratio to the dry coffee grounds from each brand to extract caffeine.
* Placed the flasks in an ultrasonic cleaner (15 W) for 10 min to facilitate caffeine dissolution.
* Collected 1 mL of the liquid from each sample and centrifuged at 15,000 g for 3 min.
* Prepared samples and performed HPLC analysis.

8.23-8.26 — Whole-Cell Catalysis of Caffeine from Dry Coffee Grounds and Extraction from Wet Coffee Grounds

* Collected coffee grounds from various stores, including Starbucks, Luckin, Kudi, 818, KFC, and Shengji.
* Diluted dry coffee grounds at a mass-to-volume ratio of 4:1 to prepare transformation solutions.

![](https://static.igem.wiki/teams/5313/notebook/notebook30.png)

* Returned to stores to collect more coffee grounds.
* Added water at a mass-to-volume ratio of 4:1 to the dry coffee grounds from each brand to extract caffeine.
* Placed the flasks in an ultrasonic cleaner (15 W) for 10 min to facilitate caffeine dissolution.
* Collected 1 mL of the liquid from each sample and centrifuged at 15,000 g for 3 min.
* Prepared samples and performed HPLC analysis.

8.27-8.29 — Whole-Cell Catalysis and Resampling of Caffeine from Coffee Grounds

* Used B12 dual transformation strain.
* Added water at a mass-to-volume ratio of 4:1 to the dry coffee grounds from each brand to extract caffeine.
* Placed the flasks in an ultrasonic cleaner (15 W) for 10 minto facilitate caffeine dissolution.
* Collected 1 mL of the liquid from each sample, centrifuged at 15,000 g for 3 min, and prepared samples (due to high levels of impurities, filtered twice through a membrane).
* Prepared samples with 5 g each of Luckin and Starbucks coffee grounds, dissolved in 20 mL, 40 mL, 80 mL, and 100 mL of water respectively, and sonicated for 10 min.
* Collected 1 mL, centrifuged at 15,000 g for 5 min, collected the supernatant, centrifuged again, and filtered twice through a 0.22μm membrane.
* Placed the flasks in an ultrasonic cleaner (15 W) for 10 minto facilitate caffeine dissolution.
* Collected 1 mL of the liquid from each sample, centrifuged at 15,000 g for 10 min, and prepared samples.

8.30-9.2 — Sample Preparation from Coffee Grounds

* Weighed 5 g of coffee grounds from each brand and dissolved them in 100 mL of water.
* Heated to boiling point five times using a microwave.
* Filtered using filter paper, collected 1 mL of the filtered liquid, and centrifuged at 15,000 g for 5 min.
* Used a 1 mL syringe to collect the centrifuged liquid and filtered it twice through a membrane.

9.3-9.9 — Production of 7-MX from Caffeine in Coffee Grounds

* Weighed 2 g of coffee grounds from each brand and dissolved them in 5 mL of Tris buffer.
* Heated to boiling point five times using a microwave.
* Filtered using filter paper, collected 1 mL of the filtered liquid.
* Centrifuged at 15,000 g for 5 min, used a 1 mL syringe to collect the centrifuged liquid, and filtered it twice through a membrane.
* From 0 h, samples were collected every 2 hfor a total period of 18 h.
* For each sampling, 50 μL of the transformation liquid was mixed with 50 μL of Tris buffer, centrifuged at 15,000 g for 10 min, and then prepared for HPLC analysis.
* Performed HPLC to produce time curves for 7-MX production from different brands of coffee grounds.

![](https://static.igem.wiki/teams/5313/notebook/notebook31.png)

![](https://static.igem.wiki/teams/5313/home/homegotop.png)

[Overview](#side1)
[1. Biosensor Construction

[First Cycle: RED homologous recombination knockout.](#side2_1)
[Second Cycle: Component Assembly](#side2_2)](#side2)
[2. Biosensor Functional Validation

[First Cycle: Preparation of the key substrate 7-MX](#side3_1)
[Second Cycle: Initial Testing of the Sensor's Substrate Specificity](#side3_2)
[Third Cycle: Second Testing of the Sensor's Substrate Specificity](#side3_3)
[Fourth Cycle: Third Testing of the Sensor's Substrate Specificity](#side3_4)
[Fifth Cycle: Sensor Sensitivity Testing](#side3_5)](#side3)
[3. Generation and Screening of Mutants](#side4)
[4. Optimization of Whole-cell catalysis Conditions

[First Cycle: Evaluation of Substrate Concentrations](#side5_1)
[Second Cycle: Bacterial Volume Concentration Gradient Experiment](#side5_2)
[Third Cycle: Time Sampling Experiments](#side5_3)](#side5)
[5. Utilizing Food Waste Coffee Grounds for 7-MX Production

[First Cycle: Initial Extraction of Caffeine](#side6_1)
[Second Cycle: Re-extraction of Caffeine](#side6_2)
[Third Cycle: Production of 7-MX Using Coffee Grounds](#side6_3)](#side6)

Engineering

Overview

![](https://static.igem.wiki/teams/5313/engineering-new/engineering-fig-01.png)

1. Biosensor Construction

First Cycle: RED homologous recombination knockout.

Design

To develop a 7-MX (7-methylxanthine)-dependent auxotrophic strain, we first analyzed the relevant metabolic pathways. Through querying the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, we identified xanthine as a downstream product of 7-MX, which is closely associated with the synthesis of DNA and RNA precursors. The survival of the strain depends on two metabolic pathways (Fig. 1): the primary pathway regulated by the *guaB* gene, and the secondary pathway utilizing xanthine as a substrate, mediated by the *gpt* gene. To render the strain dependent on 7-MX for survival, we designed a strategy to disrupt the primary metabolic pathway by knocking out the *guaB* gene, thereby activating the secondary pathway. This would force the strain to rely on xanthine metabolism, ultimately achieving the construction of the target strain.

![](https://static.igem.wiki/teams/5313/engineering-new/engineering-fig-1.png)

Fig. 1 Xanthine metabolism chart

To implement this design, we opted to use the RED homologous recombination technology to knock out the *guaB* gene and simultaneously replace it with a kanamycin resistance gene (*kana*). The introduction of the *kana* not only facilitates subsequent screening and validation, but also serves as a selectable marker, providing convenience for subsequent experiments.

Build

First, we designed and constructed the targeting fragment for homologous recombination. By retrieving genomic information, we obtained the complete sequence of the *guaB* gene. Subsequently, we amplified approximately 500 bp homologous arms upstream and downstream of the *guaB* gene using PCR. Next, we used overlap PCR to ligate the upstream and downstream homologous arms with the *kana* fragment, generatingthe targeting construct. Finally, we co-transformed this construct with the pKD46 plasmid into Escherichia coli (*E. coli*) competent cells, utilizing the RED homologous recombination system to guide the targeting fragment to recombine with the *guaB* gene locus, replacing the original *guaB* gene by *kana* gene (Fig. 2).

![](https://static.igem.wiki/teams/5313/engineering-new/engineering-fig-2.png)

Fig. 2 The *guaB* gene was knocked out and replaced by *kana* gene using the RED homologous recombination

Test

To screen for clones with successful *guaB* gene knockout, we performed colony PCR on the transformed bacteria using the primers located in the upstream and downstream homologous arms (Fig. 3). If the PCR amplification shows a 1600 bp band (corresponding to the replaced *kana* gene), it indicates successful knockout; if the result matches the negative control (i.e., a 2000 bp band, corresponding to the length of the *guaB* gene), the knockout does not occur (Fig. 3).

![](https://static.igem.wiki/teams/5313/engineering/engineering4.png)

Fig. 3 Colony PCR to screen for the knockout clones using primers located in the upper and lower homology arms.

After successfully obtaining the knockout clones, we picked up colony-PCR positive strains that were the xanthine-dependent auxotrophic strain, designated as *E. coli* BW25113-Δ*guaB*. These bacteria could survive dependently on xanthine, and this could be used as a foundation for subsequent experiments.

Learn

Through this round of experiments, we successfully constructed the xanthine-dependent strain *BW25113-ΔguaB*. This demonstrates that by knocking out the *guaB* gene, the secondary metabolic pathway was activated, making the strain depend on exogenously supplied xanthine to survive. Next, based on the caffeine metabolism pathways reported in the literature, we plan to further modify the metabolic pathways to shift the strain's dependency from xanthine to 7-MX, aiming to achieve our ultimate goal of constructing a 7-MX biosensor.

Second Cycle: Component Assembly

Design

Based on our review of previous literature[1][2][3][4], we selected the NdmDCE protein as the target enzyme for conversion. NdmDCE can effectively convert 7-MX, enabling the strain to respond to the presence of 7-MX. Therefore, we plan to introduce the exogenous NdmDCE metabolic pathway into the previously constructed *BW25113-ΔguaB* auxotrophic strain, allowing it to detect the presence and concentration of 7-MX through the expression of the NdmDCE protein. To achieve this, we designed and constructed the plasmid pYB1s-ndmDCE (pDCE).

Build

To construct pDCE, we first obtained the sequences of the *ndmD*, *ndmC*, and *ndmE* genes from the NCBI database and made them synthesized by a company. Next, we employed the Golden Gate assembly to subclone these gene fragments into the pYB1s vector (Fig. 4). After completing the plasmid construction, we introduced pDCE into the previously constructed *BW25113-ΔguaB* competent strain via chemical transformation.

![](https://static.igem.wiki/teams/5313/engineering-new/engineering-fig-4.png)

Fig. 4 Plasmid construction of pYB1s-ndmDCE

Test

To verify the successful construction of pDCE, we performed colony PCR on the transformed strains using the primers ndmC-F and ndmE-R. The experimental results showed that the length of the amplified fragment was consistent with the expected size (Fig. 5), indicating that the plasmid was successfully constructed and inserted into the target strain.

![](https://static.igem.wiki/teams/5313/engineering/engineering6.png)

Fig. 5 Results of colony PCR screening

Through further validation experiments, we confirmed the successful construction of pDCE (Fig. 6), and the target biosensor was successfully established.

![](https://static.igem.wiki/teams/5313/engineering/engineering7.png)

Fig. 6 Plasmid pYB1s-ndmDCE

Learn

Through this round of component assembly and genetic engineering, we successfully constructed a biosensor capable of detecting 7-MX. The sensor is sensitive to 7-MX concentration under optimal conditions: the strain exhibits normal growth in the presence of high concentrations of 7-MX, while its growth is inhibited under low concentration or absence 7-MX conditions. This provides a critical foundation for achieving a 7-MX-dependent sensor.

2. Biosensor Functional Validation

First Cycle: Preparation of the key substrate 7-MX

Design

To test the specificity and sensitivity of the biosensor, we need to use a large amount of 7-MX as the substrate. However, the high cost of commercially available 7-MX standards may exceed our budget. Inspired by the 2023 NEFU-China team’s project, we decided to produce 7-MX by ourselves using synthetic biology approaches [5][6].

We designed and constructed a plasmid, pYB1s-ndmDtBA (Fig. 7), which features a truncated NdmD to enhance the catalytic efficiency. This plasmid can sequentially demethylate caffeine to ultimately produce 7-MX. Additionally, we constructed a cofactor regeneration plasmid, pSB1c-frmAB-FDH (Fig. 8), which improves the production efficiency of 7-MX by reducing the accumulation of toxic by-products, which also eliminates environmental pollution. Finally, we co-transformed these two plasmids into *E. coli* BW25113 competent cells, creating a strain capable of producing 7-MX.

![](https://static.igem.wiki/teams/5313/engineering-new/engineering-fig-7.png)

Fig. 7 Plasmid construction of pYB1s-ndmDtBA

![](https://static.igem.wiki/teams/5313/engineering-new/engineering-fig-8.png)

Fig. 8 Plasmid construction of pSB1c-frmAB-FDH

Build

Construction of the 7-MX Production Pathway

We obtained the *ndmA*, *ndmB*, and *ndmD* genes through gene synthesis and amplified these fragments using PCR. We truncated the *ndmD* gene by removing the coding region for the N-terminal 266 amino acids and obtained the *ndmDt* gene fragment. Using Golden Gate technology, we replaced the original *ndmD* gene with *ndmDt*, constructing the improved pYB1s-ndmDtBA plasmid.

During the Golden Gate assembly process, the *ndmDt* gene was driven by the strong promoter pBAD, while the *ndmA* and *ndmB* genes were controlled by the J23107 promoter. Ultimately, we constructed the pYB1s-ndmDtBA plasmid (Fig. 9).

![](https://static.igem.wiki/teams/5313/engineering/engineering-fig-09.png)

Fig. 9 Plasmid pYB1s-ndmDtBA

Construction of the Cofactor Regeneration System

We amplified the frmA, frmB, and FDH genes via PCR and used DpnⅠ digestion to eliminate the template DNA, which reduced false positive clones. Subsequently, we utilized Golden Gate assembly to ligate these fragments into a plasmid, constructing the pSB1c-frmAB-FDH plasmid (Fig. 10).

![](https://static.igem.wiki/teams/5313/engineering/engineering11.png)

Fig. 10 Plasmid pSB1c-frmAB-FDH

Finally, we co-transformed the pSB1c-frmAB-FDH and pYB1s-ndmDtBA plasmids into *E. coli* BW25113 competent cells, successfully obtaining a strain capable of 7-MX production.

Test

First, we activated and induced the obtained 7-MX production strain. Then, following the designed system (Fig. 11), we added the substrate to initiate the whole-cell catalytic reaction.

![](https://static.igem.wiki/teams/5313/engineering-new/engineering-fig-11.png)

Fig. 11 Whole Cell Catalytic System Diagram

Due to the low solubility and high density of 7-MX in water, after 25 h of catalysis, we separated the bacterial cells by centrifugation at 4200 rpm for 10 min. The cells were then washed 2-3 times to extract the 7-MX precipitate. The 7-MX precipitate was subsequently dried in an oven at 60°C for 3 h, and finally ground to obtain 16 mg of 7-MX product (Fig. 12).

![](https://static.igem.wiki/teams/5313/engineering-new/engineering-fig-12a.png)
![](https://static.igem.wiki/teams/5313/engineering-new/engineering-fig-12b.png)

Fig. 12 Images of a and b (after 7-MX purification)

Learn

Through the careful design of the metabolic pathway and optimization of reaction conditions, we successfully enhanced the yield and production efficiency of 7-MX. Moreover, our production process is not only cost-effective but also environmentally friendly, which significantly reduced the generation of harmful by-products, and thereby provided a feasible solution for large-scale 7-MX production.

Second Cycle: Initial Testing of the Sensor's Substrate Specificity

Design

After successfully constructing the sensor, we needed to validate its functionality to ensure it could be effectively used in screening mutant strains. Since the sensor is based on an auxotrophic strain, its growth is significantly restricted in the absence of 7-MX or xanthine. Therefore, we designed an experimental protocol using M9 medium without xanthine, ensuring that the strain maintains limited growth due to the lack of essential substrates. We then tested the sensor's substrate specificity by adding different substrates in a 96-well plate. The growth curves of the strain were measured using a microplate reader, and the sensor strain's growth (OD600) was used to reflect its ability to utilize different substrates, thereby assessing its specificity.

Build

We selected a small quantity of commercially available methylxanthines and xanthine standards, along with the previously prepared 7-MX, as test substrates. According to the literature, most methylxanthines are water-soluble, so we dissolved seven types of methylxanthines and xanthine in water. The induced bacterial cells were washed to remove the xanthine-containing yeast extract from the ZY medium and resuspended in water. We used double-strength yeast ZY medium and LB medium to induce the sensor strain.

Next, we prepared M9 medium without yeast extract as the growth environment for the cells. A mixture of 100 μL of M9 medium, 100 μL of methylxanthine substrate solution, and 10 μL of bacterial suspension was added to a 96-well plate, and the growth curves were measured using a microplate reader.

Test

The figure below shows the changes in strain activity measured by the microplate reader over 36 h (Fig. 13).

![](https://static.igem.wiki/teams/5313/engineering/engineering14.jpg)

Fig. 13 Growth curves of the experimental bacteria in the enzyme labeling apparatus over a period of 36 h

We initially anticipated that only the strains with added 7-MX and xanthine substrates would grow well, while the growth of strains under other substrate conditions would be inhibited. However, all tested strains exhibited some degree of growth, indicating that the sensor has low specificity for the target substrate, which significantly deviates from our expected outcome.

Learn

In this round of experiments, we found that the antibiotic selection concentration may have been insufficient, leading to incomplete selection of resistant strains and, consequently, affecting the specificity performance of the sensor. After discussions with our advisor and thorough analysis, we hypothesized that the lack of strict antibiotic selection during transformation, activation, and induction may have resulted in contamination by non-target strains. This not only weakened the growth advantage of the target sensor strain but also prevented it from displaying a significant specificity advantage when cultured in the presence of target substrates (7-MX and xanthine).

To address this issue, we planned to increase the intensity of antibiotic selection in subsequent experiments and optimize the transformation and induction processes to minimize contamination. These improvements would help enhance the specificity performance of the sensor strain. We were confident that with these adjustments, the sensor's sensitivity and specificity would be significantly improved, providing a solid foundation for the subsequent screening of mutant strains.

Third Cycle: Second Testing of the Sensor's Substrate Specificity

Design

To enhance the effectiveness of antibiotic selection and ensure the acquisition of more target strains, we increased the antibiotic concentration during the experiment based on the kanamycin resistance gene in the plasmid to improve selection efficiency.

Build

We used the previously constructed 7-MX auxotrophic mutant competent strain *BW25113-ΔguaB* and transformed it with the NdmDCE plasmid. During subsequent plating, activation, and induction processes, the antibiotic concentrations were adjusted to k30 and s50(Final concentration of 30 and 50 μg/mL), thereby constructing the dual-antibiotic-resistant target strain as the improved sensor strain.

Test

To verify the feasibility of the sensor, we introduced the strain into the microplate reader along with different types of methylxanthines and xanthine, and measured the growth curves over 36 h (Fig. 14).

![](https://static.igem.wiki/teams/5313/engineering/engineering15.jpg)

Fig. 14 Growth curves of the experimental bacteria in the media containing different types of methylxanthines

Learn

After improving the selection method, the sensor's response to different types of methylxanthines and xanthine was as expected: the strain exhibited normal growth with the addition of 7-MX, while growth was inhibited with other substrates. However, we observed significant data variation in the results, which we speculate may be due to suboptimal experimental conditions, leading to a wide range of fluctuations. We plan to optimize the experimental protocol in the next round to obtain more accurate data.

Fourth Cycle: Third Testing of the Sensor's Substrate Specificity

Design

Based on previous experiments, we hypothesized that dual antibiotic selection may have resulted in excessively low OD values for the target strain. Additionally, we suspected that the low purity of the previously produced 7-MX may have contributed to significant errors. Therefore, in this round of experiments, we improved the culture medium formulation and purchased new 7-MX standards for testing.

Build

We dissolved seven types of methylxanthines and xanthine in M9 medium. During cell induction, we used double-strength yeast ZY medium and LB medium to ensure better cell quality. When transferring the cells, we washed the induced cells with water to remove the xanthine-containing yeast extract from the ZY medium, followed by washing and resuspending the cells in M9 medium. Finally, 200 μL of substrate solution and 10 μL of bacterial suspension were added to a 96-well plate for detection in the microplate reader.

Test

The experimental results are shown in the figure below (Fig. 15). To more clearly present the outcomes, we selected several key substrates related to caffeine metabolism and plotted a bar chart of the OD values of the strains over 36 h (Fig. 16).

![](https://static.igem.wiki/teams/5313/engineering-new/engineering-fig-15.jpg)

Fig. 15 Growth curves of the experimental bacteria change it according to Fig 14

![](https://static.igem.wiki/teams/5313/engineering/engineering17.png)

Fig. 16 OD value of the strain at 36 h

Learn

By comparing the results of this round with those from the third round of experiments, we observed a significant reduction in error. The sensor was able to specifically recognize the 7-MX substrate, aligning with the experimental design objectives.

Fifth Cycle: Sensor Sensitivity Testing

Design

In previous experiments, we validated the specificity of the pYB1s-ndmDCE (*ΔguaB*) sensor strain for the 7-MX substrate. This round of experiments aimed to further assess the sensor's response to different concentrations of 7-MX to test its sensitivity. We plan to vary the concentration of 7-MX and observe whether the strain's growth correlates proportionally with the substrate concentration, thereby evaluating the sensor's sensitivity.

Build

We used an improved formulation by preparing a 1 mM/L stock solution of 7-MX in M9 medium and diluting it in a gradient to concentrations ranging from 0.1 to 0.5 mM/L. After induction of the sensor strain in double-strength yeast ZY medium and LB medium, the cells were washed and resuspended to remove residual xanthine. Finally, 200 μL of each substrate concentration and 10 μL of bacterial suspension were added to a 96-well plate for detection using a microplate reader.

Test

The experimental results are shown in Fig. 17, illustrating the changes in strain growth activity over 36 h in response to varying 7-MX concentrations.

![](https://static.igem.wiki/teams/5313/engineering/engineering18.jpg)

Fig. 17 Growth curves of experimental bacteria in different concentrations of 7-MX

Learn

The experimental results indicated that the strain's growth was proportional to the 7-MX concentration, with the sensor showing a strong response to higher concentrations of 7-MX. Although sensitivity decreased under low-concentration conditions, the overall performance met expectations. We plan to further optimize the sensor design to enhance its sensitivity at lower concentrations.

3. Generation and Screening of Mutants

Design

To demethylate the position 1 and position 3 methyl groups of caffeine (1,3,7-trimethylxanthine) and convert it into the high-value compound 7-MX, which has potential applications, we designed a strategy to mutate the enzymes NdmA and NdmB. Literature indicates that the NdmA enzyme specifically recognizes caffeine and removes the position 1 methyl group, generating 3,7-dimethylxanthine, while the NdmB enzyme further removes the position 3 methyl group, producing 7-MX. However, since NdmA cannot directly generate 7-MX and NdmB cannot recognize caffeine, it is necessary to engineer the functions of these enzymes to achieve a one-step conversion of caffeine to 7-MX, thereby reducing reaction steps and the metabolic burden on the strain.

By aligning the amino acid sequences of NdmA and NdmB (Fig. 18), we observed significant homology, leading us to hypothesize that directed evolution could be used to optimize one of the enzymes to carry out demethylation at both the position 1 and position 3. We plan to perform site-directed mutagenesis on the key residues of these two enzymes and utilize the sensor module to efficiently screen for the optimal mutants, aiming to improve the production efficiency of 7-MX.

![](https://static.igem.wiki/teams/5313/engineering/engineering19.png)

Fig. 18 Comparative analysis of the amino acid sequences of NdmA and NdmB

Build

Based on an extensive review of the literature[1][7][8][9], we identified five key residues in NdmA and NdmB

(Fig. 19):

![](https://static.igem.wiki/teams/5313/engineering/engineering20.png)
![](https://static.igem.wiki/teams/5313/engineering/engineering21.png)

Fig. 19 Structureof key pocket sites of NdmA and NdmB enzymes

We plan to perform site-directed random mutagenesis on these key residues to affect the enzyme's substrate binding and catalytic efficiency. By mutating these sites, we aim to identify a mutant enzyme capable of demethylating caffeine in a single step.

The first round of screening will involve random mutagenesis at the five key residues, followed by culturing the strains and using a microplate reader to assess the mutation effects, to determine whether NdmA or NdmB has the potential for one-step 7-MX synthesis. In the second round of screening, we will refine the potential genes identified in the first round, dividing the key residues into two groups for more precise site-directed mutagenesis and screening.

In the first round of experiments, we will ligate the obtained *ndmA* and *ndmB* genes to the pYB1s-ndmDop1 plasmid and use the Golden Gate method to insert the mutated *ndmA*/*ndmB* genes into the plasmid. The sensor will then be used to detect the 7-MX production capacity of the mutant strains.

After mutating the target sites using degenerate primers, the mutated products will be amplified by PCR, and the growth of the mutant strains will be evaluated using the sensor. Strains that demonstrate robust growth indicate that the mutated gene can generate 7-MX via a single-step demethylation reaction.

Test

We conducted two rounds of mutagenesis screening as planned (Fig. 20).

![](https://static.igem.wiki/teams/5313/engineering-new/engineering-fig-20.png)

Fig. 20 The overall process of mutation screening

First Round of Screening: By performing random mutagenesis on the five key residues of NdmA and NdmB, we constructed a mutant library and used the sensor strain to screen for mutants capable of producing 7-MX. The preliminary screening results indicated that the NdmA mutants did not produce the desired effect, while a small portion of the NdmB mutants showed promising 7-MX production potential (Fig. 21).

![](https://static.igem.wiki/teams/5313/engineering-new/engineering-fig-21-ndmas.png)
![](https://static.igem.wiki/teams/5313/engineering-new/engineering-fig-21-ndmbs.png)

Fig. 21 Growth of mutant bacteria detected by microplate reader

Second Round of Screening: We divided the five key residues of NdmB into two groups based on their proximity and performed site-directed mutagenesis (Fig. 22).

![](https://static.igem.wiki/teams/5313/engineering/engineering25.png)

Fig. 22 The five key pocket residues in NdmB

Next, we introduced the mutants from both groups into 96-well plates and monitored their growth (OD values) using a microplate reader. Strains exhibiting good growth were selected for further analysis (Fig. 23).

![](https://static.igem.wiki/teams/5313/engineering-new/engineering-fig-23.png)

Fig. 23 OD600 value for 24 h of growth

Subsequently, the strains with better growth (OD > 1.5) were subjected to HPLC analysis, confirming that the product was 7-MX (Fig. 24).

![](https://static.igem.wiki/teams/5313/engineering/engineering30.png)

Fig. 24 HPLC analyses of the well-growing mutant strains

Finally, we performed mutagenesis on the key residue Gln289 and found that the strain exhibited the highest 7-MX yield when it was mutated to alanine (Fig. 24). This indicates that the Gln289 residue plays a critical role in the function of NdmB. Further saturation mutagenesis experiments confirmed that the alanine mutation produced the best results.

Learn

In this phase of the experiment, through two rounds of mutagenesis screening, we successfully validated the critical role of the Gln289 residue in NdmB, optimizing the enzyme's function to achieve a one-step caffeine demethylation reaction and significantly improving 7-MX production efficiency. This discovery lays a solid foundation for sensor-based screening and efficient 7-MX production.

Next, we will focus on optimizing the catalytic conditions of the mutant strain to further enhance the enzyme's activity and stability, aiming to achieve more efficient 7-MX synthesis in future large-scale production.

4. Optimization of Whole-cell catalysis Conditions

![](https://static.igem.wiki/teams/5313/engineering/engineering31.png)

Overview of Whole-cell catalysis condition optimization

First Cycle: Evaluation of Substrate Concentrations

Design

After screening mutants with the biosensor, we initially identified NdmBQ289A as the strain with the highest 7-MX production capability. However, considering that the screening was performed in a small-scale 96-well plate system with relatively high errors, we decided to verify its 7-MX production ability in a larger-scale experiment. We started by using a 50 OD bacterial culture for a substrate concentration gradient experiment to explore the maximum 7-MX yield.

Additionally, based on the reports that cofactor regeneration systems (CRS) can enhance the yield of wild-type strains in 7-MX production, we also investigated the functionality of the CRS in this context.

Build

According to a previous study [6][10], we determined that the optimal conditions for 7-MX production should be 25°C induction for 18 h and 20°C whole-cell catalysis for 18 h, as these conditions could best maintain enzyme activity. Therefore, our experiments to test the substrate concentration gradient was conducted as follows:

* Use a 50 OD bacterial culture.
* Centrifuge at 4200 rpm and 4°C, discard the supernatant.
* Resuspend the bacterial pellet in 1 mL of fermentation solution (Tris-HCl, pH=9) with substrate concentrations of 1 mM, 2 mM, 4 mM, 8 mM, and 10 mM.
* Perform whole-cell catalysis in a 100 mL shaking flask at 20°C on a shaker.

Test

Resuspend the bacterial pellet in 1 mL of fermentation liquid (Tris-HCl, pH=9) with substrate concentrations of 1 mM, 2 mM, 4 mM, 8 mM, and 10 mM.

![](https://static.igem.wiki/teams/5313/engineering/engineering32.png)

Fig. 25 Bar plot of the effect of different substrate concentrations on the production of experimental strain 7-MX at 50 OD without CRS system

![](https://static.igem.wiki/teams/5313/engineering/engineering33.png)

Fig. 26 Bar chart of the effects of different substrate concentrations on 7-MX production with experimental strains under 50 OD conditions

The results of our experiments to test the substrate concentration gradient are shown in Figure 25 and Figure 26. Based on these data, the strain without the CRS produced less 7-MX compared to the strain with CRS, demonstrating that the CRS still plays a crucial role in the mutant *ndmB* strain. Additionally, in the no-CRS group, the 7-MX yield decreased at a 10 mM substrate concentration compared to 8 mM, which may be due to formaldehyde accumulation, which was not efficiently removed by CRS.

Therefore, in subsequent experiments, only strains with CRS were used.

When the substrate concentration was below 4 mM, the 7-MX yield positively correlated with substrate concentrations. However, when the substrate concentration exceeded 4 mM, the 7-MX yield plateaued around the 4 mM level, while the accumulation of the intermediate product PX continued to increase.

Learn

It is evident that, with a 50 OD cell density, the maximum yield of 7-MX reaches only around 4 mM and does not increase further with higher substrate concentrations. Meanwhile, caffeine is still nearly completely converted, and the accumulation of PX continues to rise. We cannot rule out the possibility that relatively low cell density is a limiting factor. Therefore, the next step will be to conduct a cell density gradient experiment to investigate whether increasing the cell density can break the bottleneck of 7-MX production.

Second Cycle: Bacterial Volume Concentration Gradient Experiment

Design

Based on the results from the first round of optimization, we decided to conduct a cell density gradient experiment using the NdmBQ289A mutant under the highest substrate concentration (10 mM) to explore the condition of maximum 7-MX production.

Build

Based on existing research and the results from the first round, we decided to induce the bacteria at 25°C for 18 h.

For the whole-cell catalysis, the gradient experiment groups were set as follows: 10 OD, 20 OD, 40 OD, 80 OD, and 100 OD.

All other conditions were consistent: After centrifugation at 4200 rpm and 4°C for 10 min, the supernatant was discarded. The cell pellets were resuspended in 1 mL of fermentation solution (Tris-HCl, pH=9, 10 mM substrate) in a 10 mL centrifuge tube.

Finally, the whole-cell catalysis was conducted in a 100 mL shaking flask at 20°C and 200 rpm for 18 h.

Test

![](https://static.igem.wiki/teams/5313/engineering/engineering34.png)

Fig. 27 The bar chart of the effects of different OD value on 7-MX production with experimental strains cultured in medium containing 10 mM caffeine

The experiment of gradient cell biomass yielded the results in Figure 27. Based on the data, with increased cell biomass, especially beyond 50 OD, the yield of 7-MX was significantly improved. During this process, the consumption of the substrate caffeine gradually reached 100%. Under the 100 OD condition, the concentration of the intermediate product PX was approximately 1 mM, and the molar conversion rate of 7-MX was enhanced to about 90%.

Learn

As shown by the experimental results, under the conditions of 100 OD cell biomass and 10 mM substrate concentration, the yield of 7-MX reached a peak of 9 mM, representing a significant breakthrough. Caffeine was completely converted, and the accumulation of PX was minimized under optimal conditions. From the results of these two rounds of experiments, it is evident that even at a 50 OD cell biomass, caffeine consistently achieves a relatively high molar conversion rate, while the intermediate product PX continues to accumulate. This likely indicates that the conversion of intermediate PX to 7-MX remains a rate-limiting step even when using NdmB Q298A for 7-MX production. To confirm this hypothesis, we plan to perform time-course sampling during the whole-cell catalysis process.

Third Cycle: Time Sampling Experiments

Design

Based on previous research results and the observed accumulation of intermediate product PX, we hypothesize that the conversion of intermediate PX to the product 7-MX is the primary rate-limiting step. We have decided to perform time-course sampling during the whole-cell catalysis to validate this hypothesis.

Build

Based on existing research and the results from the first two rounds, we have determined that the highest conversion rate can be achieved with a biomass of 100 OD at a substrate concentration of 10 mM. Therefore, we decided to perform time-course sampling under these conditions.

Other conditions remain consistent: induction at 25°C for 18 h. After centrifugation at 4200 rpm and 4°C, the supernatant was discarded, and the bacterial pellet was resuspended in 1 mL fermentation broth (Tris-HCl, pH=9, 10 mM substrate) in a 10 mL centrifuge tube. Finally, whole-cell catalysis was carried out in a 100 mL shaker flask at 20°C and 200 rpm.

During the whole-cell catalysis, samples were collected every hour.

Test

![](https://static.igem.wiki/teams/5313/engineering/engineering35.png)

Fig. 28 Time curve of 7-MX production with the experimental strains cultured in medium containing 10 mM caffeine with bacteria in 50 OD

The relationship between catalysis time and the amounts of each substance is shown in Figure 28. During the 18-h whole-cell catalysis, the substrate is nearly completely converted within the first 4 h. The remaining time is for the slow conversion of the intermediate product PX to 7-MX, with the conversion rate decreasing over time.

Learn

By plotting the catalytic time curve, we confirmed the previous hypothesis that the conversion efficiency of PX to 7-MX is relatively slow, and the overall conversion rate decreased over time. Nevertheless, under conditions of 100 OD cell density and 10 mM substrate concentration, we achieved the optimal yield of 7-MX, laying the foundation for further practical production applications. Next, we will utilize these optimized conditions to attempt caffeine extraction from spent coffee grounds and produce 7-MX, aiming to recycle food waste and explore more economically viable production strategies.

5. Utilizing Food Waste Coffee Grounds for 7-MX Production

First Cycle: Initial Extraction of Caffeine

Design

Before converting caffeine in coffee grounds to 7-MX, we plan to first determine the caffeine content in the coffee grounds and preliminarily estimate the potential yield of 7-MX.

Build

We first collected sufficient coffee grounds from four coffee shops: Luckin, Starbucks, Kudi, and 818, and then dried them in an oven. After that, we performed gradient extraction of caffeine from the coffee grounds obtained from Luckin and Starbucks. We took 5 g of dry coffee grounds and dissolved them in 20 mL, 40 mL, 80 mL, and 100 mL of distilled water. The flasks were then placed in an ultrasonic bath for 10 min to enhance caffeine dissolution. After dissolution, 1 mL of the liquid was transferred to a 1.5 mL EP tube and centrifuged at 15,000 g for 5 min. The supernatant was collected, centrifuged again, and then passed through a filter using a 1 mL syringe. The filtered liquid was transferred to a brown liquid phase bottle. The prepared samples were then subjected to HPLC analysis to determine the caffeine content.

![](https://static.igem.wiki/teams/5313/engineering/engineering36.png)

Fig. 29 Different brands of collected coffee grounds

Test

![](https://static.igem.wiki/teams/5313/engineering/engineering37.png)
![](https://static.igem.wiki/teams/5313/engineering/engineering38.png)

Fig. 30 Bar chart of the concentration of caffeine extracted from Luckin and Starbucks coffee grounds

Our first-round testing results showed that the caffeine content in coffee grounds from both brands ranged between 90 mg and 100 mg per gram, which is significantly different from the theoretical values of 0.5% to 1.5% found in existing literature[11]. Therefore, we conclude that this test was not successful.

Learn

We suspect that the high caffeine content measured by us may be due to the use of ultrasonic dissolution, which could have caused the breakdown of certain substances in the coffee (such as lipids and proteins), leading to inflated measurements. Therefore, we decided to switch to heating and boiling to facilitate caffeine dissolution. Additionally, considering that a low dilution factor might result in insufficient dissolution of caffeine from the coffee grounds, we plan to increase the dilution factor to further enhance the dissolution capability.

Second Cycle: Re-extraction of Caffeine

Design

Based on the results from the previous round, ultrasonic-assisted caffeine dissolution was ineffective. Therefore, we changed our method and opted for heating and boiling to promote the dissolution of caffeine.

Build

We took 5 g of dry coffee grounds from each store and added them to 100 mL of distilled water. The mixture was heated in a microwave oven to boil 5 times, allowing the caffeine to fully dissolve into the distilled water. After cooling, the solution was filtered using a funnel and filter paper to remove the residue, leaving the filtrate. We then transferred 1 mL of the filtrate into a 1.5 mL EP tube, centrifuged it at 15,000 g for 5 min, and collected the supernatant with a 1 mL syringe. The supernatant was filtered twice through membranes and then transferred to a brown liquid chromatography bottle. The sample was prepared for subsequent analysis by HPLC to determine the caffeine content.

![](https://static.igem.wiki/teams/5313/engineering-new/engineering-fig-31.png)

Fig. 31 The overall process of extracting caffeine from coffee grounds

Test

![](https://static.igem.wiki/teams/5313/engineering/engineering40.png)

Fig. 32 Bar chart of the concentration of caffeine extracted from 4 different brands

With the improved method, the caffeine content extracted from the coffee grounds of the four brands ranged from 5 mg/g to 10 mg/g, corresponding to 0.5% to 1% of the dry weight of the coffee grounds. This is consistent with the previously reported values of 0.5% to 1.5%. Therefore, we have successfully determined the caffeine content in the coffee grounds from the four brands, and our testing results are accurate.

Learn

We have recognized the drawbacks of ultrasonic extraction. In future experiments, we will first consider whether the method might interfere with the substances being tested before deciding to use it.

Third Cycle: Production of 7-MX Using Coffee Grounds

Design

Attempt to Use Coffee Grounds as a Low-Cost Raw Material: We plan to explore the use of coffee grounds as a raw material for producing 7-MX, as this approach reduces the costs and offers a new method for recycling coffee grounds. By utilizing our optimized strains, we aim to produce 7-MX from the caffeine in coffee grounds while evaluating both the environmental and economic benefits. We will use the optimized strains to perform whole-cell catalysis with conversion solutions derived from coffee grounds of various brands and plot the corresponding time curves.

Build

We plan to use the optimized NdmBQ289A strain for the production of 7-MX. We will first prepare conversion solutions by extracting caffeine. For each brand of coffee grounds, 2 g will be weighed and dissolved in 5 mL of Tris buffer. The mixture will be heated to boiling at 100°C in a water bath for 10 min to ensure complete dissolution of caffeine. After cooling, the solution will be centrifuged at 4000 rpm for 10 min at 4°C. The supernatant will be transferred to a 2 mL EP tube and centrifuged again at 15000 g for 10 min. The supernatant will be used as the conversion solution.

The induced NdmBQ289A strain will be used for whole-cell catalysis. We will use 100 OD of the induced strain, resuspend it in 1 mL of the conversion solution, and place it in a 100 mL Erlenmeyer flask. Whole-cell catalysis will be conducted under the optimized conditions for 18 h. Starting from 0 h, a 50 μL aliquots will be taken every 2 h, diluted with 50 μL of Tris buffer, and centrifuged at 15000 g for 3 min. The supernatant will be sampled and analyzed by HPLC to determine the 7-MX content and calculate the conversion rate.

![](https://static.igem.wiki/teams/5313/engineering/engineering41.png)

Fig. 33 Procedures before 18 h fermentation

![](https://static.igem.wiki/teams/5313/engineering/engineering42.png)

Fig. 34 Mix the NdmBQ289A strain with transformation liquid containing caffeine

Test

![](https://static.igem.wiki/teams/5313/engineering/engineering43.png)

Fig. 35 The fermentation curves with 100 OD NdmBQ289A over 18 h

The results show that at 0 h, only caffeine was present, with no 7-MX detected. As the fermentation progressed, the caffeine content gradually decreased, while the levels of 7-MX and PX increased until the caffeine was depleted. We conclude that caffeine was successfully converted to the target product, 7-MX, indicating that our test was successful. However, compared to fermentation with pure caffeine, the conversion rate of 7-MX was not ideal. We speculate that some substances in the coffee grounds may have interfered with the enzyme's activity, reducing the conversion rate of 7-MX.

Learn

As shown by the experimental results， we can successfully utilize caffeine from spent coffee grounds for the efficient production of 7-MX, making a significant advancement in the project. The experiment not only showed successful conversion of caffeine into 7-MX but also highlighted the robust conversion capability of our optimized NdmBQ289A strain in practical applications. Although the conversion rate of 7-MX from spent coffee grounds was slightly lower compared to pure caffeine, this result confirmed that we have achieved the goal of producing high-value compounds from waste materials.

Reference:

[1] Kim JH, Kim BH, Brooks S, Kang SY, Summers RM, Song HK. Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex. J Mol Biol. 2019 Sep 6;431(19):3647-3661. doi: 10.1016/j.jmb.2019.08.004. Epub 2019 Aug 11. PMID: 31412262.

[2] Summers RM, Louie TM, Yu CL, Gakhar L, Louie KC, Subramanian M. Novel, highly specific N-demethylases enable bacteria to live on caffeine and related purine alkaloids. J Bacteriol. 2012 Apr;194(8):2041-9. doi: 10.1128/JB.06637-11. Epub 2012 Feb 10. PMID: 22328667; PMCID: PMC3318484.

[3] Mock MB, Summers RM. Mixed culture biocatalytic production of the high-value biochemical 7-methylxanthine. J Biol Eng. 2023 Jan 10;17(1):2. doi: 10.1186/s13036-022-00316-6. PMID: 36627657; PMCID: PMC9830774.

[4] Algharrawi KH, Summers RM, Gopishetty S, Subramanian M. Direct conversion of theophylline to 3-methylxanthine by metabolically engineered *E. coli*. Microb Cell Fact. 2015 Dec 21;14:203. doi: 10.1186/s12934-015-0395-1. PMID: 26691652; PMCID: PMC4687300.

[5] Algharrawi, K.H., & Subramanian, M.V. (2020). Production of 7-methylxanthine from Theobromine by Metabolically Engineered *E. coli*. Journal of Chemical and Petroleum Engineering, 21, 19-27.

[6] Liu, C., Wu, Y., Zhao, H., Gu, X., Gu, J., Zhao, M., Zuo, S., & Wang, P. (2024). Highly Efficient Whole-Cell Biocatalysis for the Biosynthesis of 7-Methylxanthine and Other Xanthine Derivatives. ACS Sustainable Chemistry & Engineering.

[7] Liu J, Tian J, Perry C, Lukowski AL, Doukov TI, Narayan ARH, Bridwell-Rabb J. Design principles for site-selective hydroxylation by a Rieske oxygenase. Nat Commun. 2022 Jan 11;13(1):255. doi: 10.1038/s41467-021-27822-3. PMID: 35017498; PMCID: PMC8752792.

[8] Tian J, Liu J, Knapp M, Donnan PH, Boggs DG, Bridwell-Rabb J. Custom tuning of Rieske oxygenase reactivity. Nat Commun. 2023 Sep 20;14(1):5858. doi: 10.1038/s41467-023-41428-x. PMID: 37730711; PMCID: PMC10511449.

[9] Brimberry M, Garcia AA, Liu J, Tian J, Bridwell-Rabb J. Engineering Rieske oxygenase activity one piece at a time. Curr Opin Chem Biol. 2023 Feb;72:102227. doi: 10.1016/j.cbpa.2022.102227. Epub 2022 Nov 18. PMID: 36410250; PMCID: PMC9939785.

[10] Zhou B, Ma C, Ren X, Xia T, Li X. LC-MS/MS-based metabolomic analysis of caffeine-degrading fungus Aspergillus sydowii during tea fermentation. J Food Sci. 2020 Feb;85(2):477-485. doi: 10.1111/1750-3841.15015. Epub 2020 Jan 6. PMID: 31905425.

[11] Vandeponseele A, Draye M, Piot C, Chatel G. Study of Influential Parameters of the Caffeine Extraction from Spent Coffee Grounds: From Brewing Coffee Method to the Waste Treatment Conditions. Clean Technologies.2021;3(2):335-350. https://doi.org/10.3390/cleantechnol3020019

![](https://static.igem.wiki/teams/5313/home/homegotop.png)

[Directory](#side1)
[RED Homologous Recombination](#side2)
[Plasmid Preparation](#side3)
[Transformation](#side4)
[PCR](#side5)
[SDS-PAGE](#side6)
[Preparation of Competent Cells](#side7)
[Preparation of Competent Cells for Chemical Transformation](#side8)
[Gel Preparation](#side9)
[Goldengate](#side10)
[Colony PCR](#side11)
[Restriction Digestion](#side12)
[Microplate Reader Detection](#side13)
[Digestion](#side14)
[Enzyme Assay](#side15)
[Induction](#side16)
[Whole-Cell Catalysis](#side17)
[Sample Preparation](#side18)
[Coffee Grounds Collection and Drying](#side19)
[Caffeine Extraction and Detection from Coffee Grounds](#side20)
[Attempt to Produce 7-MX Using Coffee Grounds](#side21)

Experiment

Directory

1. RED Homologous Recombination

2. Plasmid Preparation

3. Transformation：Electroporation Procedure,Chemical Transformation

4. PCR

5. SDS-PAGE

6. Preparation of Competent Cells

7. Preparation of Competent Cells for Chemical Transformation

8. Gel Preparation

9. Goldengate

10. Colony PCR

11. Restriction Digestion

12. Microplate Reader Detection

13. Digestion

14. Enzyme Assay

15. Induction

16. Whole-Cell Catalysis

17. Sample Preparation

18. Coffee Grounds Collection and Drying : Coffee Grounds Collection, Coffee Grounds Drying.

19. Caffeine Extraction and Detection from Coffee Grounds: Caffeine Extraction, Sample Preparation, HPLC Detection

20. Attempt to Produce 7-MX Using Coffee Grounds :Extraction of Caffeine as a Production Substrate,Production of 7-MX,Sample Preparation for 7-MX Detection,HPLC Detection

RED Homologous Recombination

1. Design Homologous Arms and Knockout Fragment: To knockout the target gene, we choose an antibiotic resistance gene (e.g., kanamycin or chloramphenicol resistance gene) as a selection marker. Flank this resistance gene with sequences homologous to the upstream and downstream regions of the target gene (typically about 500 bp on each side). These flanking regions are used as homologous arms.

2. PCR Amplification of Knockout Fragment: Use a plasmid containing the resistance gene (e.g., pKD3 or pKD46) as a template for PCR amplification of the resistance gene fragment with homologous arms.

3. Transformation of Resistance Fragment: Introduce the PCR-amplified resistance gene fragment into bacteria that already carry the λ-RED system via electroporation. Prior to this, induce the expression of the recombination genes in the λ-RED system using arabinose to enhance recombination efficiency.

4. Homologous Recombination: Under the action of the λ-RED system, the exogenous resistance gene fragment will replace the target gene in the bacterial chromosome through homologous recombination, thereby achieving gene knockout.

5. Screening and Verification: Plate the transformed bacteria on selective media containing the appropriate antibiotic to select for resistant colonies. Extract genomic DNA from resistant colonies and verify the gene knockout by PCR or sequencing.

Plasmid Preparation

1. Harvest Cells: Transfer the overnight cultured medium to a 1.5 mL EP tube using a pipette. Centrifuge at 10,000×g for 1 min to collect the cell pellet. Discard the supernatant. Repeat this step to enrich the cell pellet.

2. Resuspend Cells: Add 250 μL of Solution I (containing RNase A and stored at 4°C) to the cell pellet. Use a pipette to resuspend the cells until no clumps are visible.

3. Lysis: Add 250 μL of Solution II and gently invert the tube 4-7 times. Incubate for 2-3 min to lyse the cells.

4. Neutralization: Add 350 μL of Solution III and gently invert the tube 4-7 times. Centrifuge at maximum speed (15,000×g) for 10-15 min.

5. Bind DNA: Carefully transfer the supernatant to a blue adsorption column and centrifuge at maximum speed for 1 min. Discard the waste liquid and return the column to an empty collection tube.

6. Wash: Add 500 μL of HBC Buffer (containing isopropanol) to the adsorption column. Centrifuge at maximum speed for 1 min, discard the waste liquid, and return the column to an empty collection tube.

7. Wash Again: Add 700 μL of DNA Wash Buffer to the adsorption column. Centrifuge at maximum speed for 1 min, discard the waste liquid, and repeat this step twice.

8. Dry: Place the adsorption column in an empty collection tube and centrifuge at maximum speed for 2 min to remove any residual wash buffer.

9. Elute DNA: Transfer the adsorption column to a clean 1.5 mL EP tube. Place the tube in a metal bath at approximately 55°C for 3-5 min to dry.

10. Elution: Add 30-50 μL of distilled water to the center of the adsorption membrane. Let it stand at room temperature for 1 min, then centrifuge at maximum speed for 1 min to elute the DNA.

11. Storage: Store the extracted DNA at -20°C.

Transformation

Electroporation Procedure

1. Add the target fragment (greater than 200 ng, preferably not exceeding 5 mL) to 100 mL of competent cells and place on ice for 10-30 min.

2. Sterilize and dry 2 mm electroporation cuvettes under UV in a laminar flow hood for 20 min, pre-chill on ice, and quickly transfer the competent cells to the cuvette, ensuring cells are at the bottom.

3. Perform the following steps quickly: Wipe the outside of the cuvettes dry. For 2 mm cuvettes, use electroporation program Ec2; for 1 mm cuvettes, use program Ec1. After electroporation, immediately add 900-1000 µL of pre-warmed LB at 37°C, gently pipette, and transfer to a 1.5 mL centrifuge tube. Incubate at 30°C on a shaking incubator at 150 rpm for 45-60 min.

4. Plate an appropriate volume onto Kana+Str double antibiotic plates and incubate at 37°C.

Chemical Transformation

1. Thaw competent cells (stored in a -80°C freezer) on ice, add the ligation product/plasmid, and incubate on ice for 20 min.

2. Heat shock at 42°C for 45 sec, then let the cells rest on ice for 2 min.

3. Add 600 µL of LB medium without antibiotics and incubate at 37°C with shaking for 45 min.

4. Centrifuge at 4000 rpm for 2 min, discard an appropriate amount of supernatant, and plate the cells (using both diluted and concentrated samples if needed). Label the plates accordingly.

5. Incubate the plates upside down at 37°C for 12 h.

PCR

1. Prepare the reaction mixture in a PCR tube on ice and mix thoroughly using a vortex mixer.

2. Collect any residual liquid on the tube walls using a mini centrifuge.

3. Transfer the PCR tube to the thermal cycler and set the program to initiate the PCR thermal cycling process.

![](https://static.igem.wiki/teams/5313/experiment/experiment1.png)

SDS-PAGE

1. Gel preparation:

Assemble the module and secure the gel mold. Prepare the lower gel according to the following recipe, pour it into the mold, and add water to level the surface. Let it sit for 40-60 min until the gel solidifies. Once solidified, discard the water on the surface. Prepare the upper gel according to the recipe, pour it into the mold, insert the comb, and allow it to solidify for approximately 1 h. After solidification, dismantle the mold to complete the gel preparation.

2. Sample preparation:

Collect the induced and uninduced bacterial cultures, measure and obtain 6 OD units of bacterial suspension. Centrifuge at 10,000 g for 10 min, discard the supernatant, and resuspend the pellet in 600 µL of distilled water. Use an ultrasonic cell disruptor to lyse the cells. Centrifuge the lysate at 10,000 g for 10 min to separate the supernatant and pellet. Both fractions are heated at 100°C in a metal bath for 5 min for inactivation, completing the sample preparation.

3. Sample loading:

Prepare the protein buffer solution according to the table above. Place the gel into the electrophoresis apparatus. After checking for leaks with the buffer solution, mix 16 µL of the prepared sample with 4 µL of loading buffer. Load 16 µL of the mixture into the wells of the gel, followed by 4 µL of the protein marker. Then, sample loading is complete.

4. Electrophoresis:

Connect the power supply and run the gel at 80 V for 30 min, followed by 220 V for about 1 h.

5. Staining:

Carefully peel the gel off in water, then place the gel into the staining solution. Boil the solution and then use the heated staining solution to stain the gel at room temperature for 1 h.

6. Destaining:

Place the stained gel in boiling destaining solution and destain it three times.

Composition:

Upper gel:

![](https://static.igem.wiki/teams/5313/experiment/experiment22.png)

Lower Gel :

![](https://static.igem.wiki/teams/5313/experiment/experiment34.png)

Protein Destaining Solution (500 mL system):

Anhydrous ethanol (75 mL), acetic acid (50 mL), distilled water (375 mL).

5× Protein Buffer Solution (5× Tris-Glycine Buffer; 1 L system):

Tris base (15.1 g), glycine (94 g), SDS (5 g); adjust the pH to 8.3 with precise measurements.

SDS-PAGE Staining Solution (400 mL system):

Coomassie Brilliant Blue R-250 (0.4 g), isopropanol (100 mL), glacial acetic acid (40 mL), distilled water (260 mL); stir thoroughly and filter using filter paper, frequently replacing the paper to remove particulate matter. Prepare two bottles at once, which can be reused.

5×Protein SDS-PAGE Loading Buffer:

(Finally, the volume of distilled water was fixed to 5 mL, and stored at -20 ℃.)

Preparation of Competent Cells

1. Transfer 200 μL of the glycerol stock into 5 mL of LB liquid medium and culture at 30°C, 200 rpm for approximately 12 h. Subsequently, inoculate into LB medium at a 2% concentration and incubate for 0.5 h before adding arabinose to a final concentration of 0.2%.

![](https://static.igem.wiki/teams/5313/experiment/experiment33.png)

2. After culturing at 30°C for approximately 2 to 2.5 h, achieving an OD₆₀₀ of about 0.55-0.6 (values significantly higher or lower can severely affect electroporation efficiency), remove the culture and incubate on ice for 30 min.

3. Pre-cool the centrifuge rotor in a freezer for 10 min. Collect the cells by centrifugation using pre-cooled centrifuge tubes and keep on ice. Centrifuge at 4000 rpm for 10 min, aliquoting 50 mL per tube.

4. Using 18 mL of pre-cooled, sterile 10% glycerol per tube, gently pipette while keeping the tube on ice. Aliquot into two 10 mL centrifuge tubes and centrifuge at 4200 rpm for 10 min in a low-temperature centrifuge.

5. Immediately after centrifugation, discard the supernatant. Add pre-cooled, sterile 10% glycerol and then pipette gently while keeping the tube on ice.

6. Repeat steps 4 and 5 three times. For the first two washes, use 5 mL of pre-cooled, sterile 10% glycerol. For the final wash, resuspend in 0.25 mL of 10% glycerol and combine the two tubes into one.

7. Aliquot 100 μL per tube into pre-cooled centrifuge tubes. Proceed with electroporation or store immediately at -80°C. The competent cells can be stored at -80°C for up to six months.

Preparation of Competent Cells for Chemical Transformation

1. From -20°C or -80°C glycerol stock, use a sterile loop or white pipette tip to pick up the bacterial culture. Streak it onto an agar plate and incubate for 12 h (to isolate single colonies).

2. Pick single colonies and inoculate into 5 mL of liquid LB medium using a white pipette tip (or by pipetting up and down). Label the tubes and incubate at 37°C with shaking for 12 h.

3. Transfer 1 mL of the LB culture into 100 mL of LB medium and incubate at 37°C with shaking for about 2 h. Measure the OD until it reaches approximately 0.4 (monitor time carefully).

4. Place the culture into a 16°C shaker and incubate for about 1 huntil the OD reaches 0.5.

5. At this stage, do not use the alcohol lamp. Perform all procedures under strict aseptic conditions on ice. Aliquot 100 mL of the culture into four 50 mL centrifuge tubes and let sit on ice for 30 min to stop active growth.

6. Centrifuge at 4°C and 4200 rpm for 10 min. After centrifugation, place the tubes on ice, quickly discard the supernatant, and remove residual liquid using a pre-cooled pipette tip. Add 1 mL of a mixed solution of 80 mM MgCl₂ and 20 mM CaCl₂ to each tube (you may combine centrifuge tubes, ensuring balance).

7. Incubate on ice for 30 min.

8. Centrifuge at 4°C and 4200 rpm for 10 min. After centrifugation, place the tubes on ice, quickly discard the supernatant using aseptic techniques, and remove residual liquid with a pre-cooled pipette tip. Resuspend the pellet in 4 mL of a 15% glycerol and 100 mM CaCl₂ mixture, gently shaking on ice.

9. Aliquot 100 μL into pre-cooled tubes. Record the number of aliquots prepared. Prepare labeling for the storage bags, including preparation date, preparer, competent cell type, and aliquot volume. Place the labeling bags in ice for cooling.

10. Store at -80°C.

Gel Preparation:

1. Prepare agarose gels of appropriate concentration: 1% for detection gels and 2% for recovery gels. Heat in a microwave until the agarose is fully dissolved.

2. Allow the solution to cool to approximately 60°C, then add 1/10,000 Goldview and mix thoroughly.

3. Place an appropriately sized comb into the gel mold. Pour the warm agarose gel into the mold, ensuring the gel thickness is between 3-5 mm. Allow the gel to solidify for approximately 20 min.

4. Once the gel has completely solidified, gently remove the comb and place the gel tray into the electrophoresis chamber. Add 1×TAE buffer so that the buffer level is about 1 mm above the gel surface, ensuring proper conductivity.

Sample Loading:

1. Add the following to the wells: (PCR products + loading buffer) or DNA marker.

Electrophoresis:

1. Cover the electrophoresis chamber and run the gel at 80-120V.

2. When the dye front has migrated an appropriate distance through the gel (approximately 30 min), turn off the power, remove the gel, and observe the DNA bands under a UV lamp.

Gel Imaging:

1. Place the gel onto the gel imaging system and capture an image under ultraviolet light to visualize the gel.

Goldengate

1. Design primers with restriction enzyme recognition and cleavage sites added to both ends of the vector and the target gene fragment.

2. Prepare the reaction system in PCR tubes on ice, and mix thoroughly.Centrifuge to collect the liquid at the bottom of the tube.

![](https://static.igem.wiki/teams/5313/experiment/experiment4.png)

3. Centrifuge to collect the liquid at the bottom of the tube.

4. Transfer the PCR tubes to the thermal cycler and set the parameters to initiate the ligation.

![](https://static.igem.wiki/teams/5313/experiment/experiment5.png)

Colony PCR

1. Prepare the colony PCR reaction system. For a 10 µL reaction volume, use: 1 µL of bacterial suspension, 0.4 µL of upstream and downstream primers, 5 µL of PCR Mix, and 3.2 µL of ddH₂O. If directly picking a colony, add ddH₂O to a final volume of 10 µL. For a 50 µL reaction volume, scale up the components proportionally

2. Set up the PCR program according to the length of the bacterial PCR fragment and run the PCR.

3. Prepare the electrophoresis gel based on the number of bacterial PCR products and the system.

4. Load the bacterial PCR products and an appropriately sized DNA marker into the wells of the gel. Place the gel into the electrophoresis chamber and run the gel at 120-180V.

5. Determine the electrophoresis completion time based on the marker bands. After completion, remove the gel, visualize under UV light, and photograph for analysis.

Restriction Digestion

1. Select appropriate restriction sites based on the plasmid map.

2. Identify the corresponding restriction enzymes and system instructions, and prepare the reaction mixture according to the instructions.

3. Incubate the prepared mixture at the specified temperature in a metal bath or PCR machine for the recommended time.

Microplate Reader Detection

1. The strain was washed 3 times with water and then washed with M9 medium 2 times to finally obtain the bacterial solution dissolved in M9.

2. Multi-well plate preparation: Sterilize and label the 96-well plate, and add 200 μL of aqueous solutions of different solvents to be tested to the wells.

3. Add 200 μL of bacterial solution dissolved in M9 to each well.

4. Cultivate under suitable conditions.

5. Set the corresponding parameters on the microplate reader to measure the growth of each well strain.

Digestion

1. Add Dpn I enzyme: Add 1 µL of Dpn I enzyme (10 U/µL) to each 50 µL PCR reaction mixture.

2. Gently mix the reaction mixture to avoid vigorous shaking and incubate at 37°C for 1 h to ensure complete digestion of the methylated template plasmid by Dpn I.

3. After digestion is complete, proceed with bacterial transformation without the need to deactivate Dpn I.

![](https://static.igem.wiki/teams/5313/experiment/experiment6.png)

Enzyme Assay

1. Wash the bacterial culture three times with water and then wash twice with M9 medium to obtain the final bacterial suspension in M9 medium.

2. Prepare a 96-well plate: sterilize and label the plate, and add 200 µL of different solvents and different concentrations of aqueous solutions to the wells.

3. Add 200 µL of the bacterial suspension in M9 medium to each well.

4. Incubate under appropriate conditions.

5. Set the corresponding parameters on the plate reader and measure the growth of the bacteria in each well.

Induction

1. Turn on the UV light in the laminar flow hood and sterilize for 10-20 min.

2. Turn off the UV light, then turn on the fan and lighting. Sterilize and place the required glycerol stocks, antibiotics, and LB medium into the laminar flow hood.

3. Add 5 µL of the antibiotic corresponding to the bacterial strain (the volume of antibiotic added should be 1/1000 of the medium volume) to 5 mL of LB medium in a test tube.

4. Transfer 50 µL of bacterial culture from the glycerol stock (the volume of glycerol stock added should be 1/100 of the medium volume) into the LB medium with the added antibiotic.

5. Turn off the fan and lighting, remove the test tube, and incubate at 37°C with shaking for 12 h.

6. After incubation, prepare ZY medium (shaking flask), 50×M, 50×5052, trace elements, MgSO₄, and antibiotics, and sterilize them before placing them into the sterilized laminar flow hood.

7. Add the following to the ZY medium (shaking flask) using a pipette: 2 mL of 50×M, 2 mL of 50×5052, 200 µL of trace elements, 200 µL of MgSO₄, 100 µL of the corresponding antibiotic, and 1 mL of the bacterial culture. Seal the flask with parafilm and secure it with a rubber band.

8. Remove the flask and incubate at 37°C with shaking for 1-2 h until the OD600 reaches 0.5.

9. After incubation, prepare 20% arabinose and IPTG as needed, sterilize them, and place them into the sterilized laminar flow hood.

10. Add 1 mL of 20% arabinose (final concentration 0.2%) and 100 µL of IPTG to the ZY medium (shaking flask) using a pipette. Seal the flask with parafilm and secure it with a rubber band.

11. Remove the flask and incubate at 25°C with shaking for 18 h.(Fig. 1)

![](https://static.igem.wiki/teams/5313/experiment/experiment7.png)

Fig. 1 Flasks for incubation at 25°C

Whole-Cell Catalysis

1. Remove the induced shaking flask, take the bacterial culture with the corresponding OD value, and transfer it to a 10 mL centrifuge tube. Centrifuge at 4200 rpm and 4°C for 10 min.

2. Discard the supernatant and add 1 mL of Tris-HCl (pH=9) containing the appropriate substrate concentration of caffeine to the pellet. Mix thoroughly.

3. Transfer the mixed solution to a 100 mL shaking flask.(Fig. 2)

![](https://static.igem.wiki/teams/5313/experiment/experiment8.png)

Fig. 2 Whole-cell catalyzed sample vials made

4. Incubate the shaking flask at 20°C with shaking for 18 h.(Fig. 3)

![](https://static.igem.wiki/teams/5313/experiment/experiment9.jpg)

Fig. 3 Thermal flasks for incubation at 20°C

Sample Preparation

1. Use a pipette to add the appropriate amount of Tris-HCl (pH=9) to the shaking flask to dilute the solution (dilution factor is substrate concentration/2) and mix thoroughly.

2. Transfer 1 mL of the bacterial culture from the shaking flask to an EP tube and centrifuge at 10,000×g for 10 min.

3. After centrifugation, remove the EP tube and use a 1 mL syringe to aspirate the entire supernatant.

4. Attach the syringe to a 0.22 µm filter membrane, and push the syringe to filter the supernatant into a new EP tube. Repeat the process if necessary to ensure complete filtration.

5. Use a pipette to transfer the filtered supernatant into a brown liquid-phase vial. The sample is now prepared and can be stored at -20°C for short-term use.

Coffee Grounds Collection and Drying

Coffee Grounds Collection

1. Search for nearby coffee shops and prepare to request coffee grounds.

2. Identify four coffee shops: Ruixing, Starbucks, Kudi, and 818.

3. Organize a team to visit the shops and request coffee grounds from the staff.

Coffee Grounds Drying

1. Spread the coffee grounds from each brand separately in foam boxes.

2. Place the foam boxes in an oven set to 60°C and dry for 48 h.

Caffeine Extraction and Detection from Coffee Grounds

Caffeine Extraction

1. Weigh 5 g of dried coffee grounds and add 100 mL of distilled water. Shake to ensure complete dissolution.

2. Heat the mixture in a microwave until boiling, and allow it to boil for 5 cycles.

3. After cooling, filter the solution using a funnel and filter paper to remove undissolved residues, retaining the filtrate.

4. Transfer 1 mL of the filtrate to a 1.5 mL EP tube using a pipette. Place the EP tube in a centrifuge and set to 15,000 g for 5 min.

Sample Preparation

1. After centrifugation, remove the 1.5 mL EP tube and use a 1 mL syringe to aspirate all of the supernatant.

2. Attach a yellow filter membrane to the 1 mL syringe and filter the liquid into a new 1.5 mL EP tube. Repeat the process to ensure complete filtration.

3. Use a 1 mL syringe to aspirate the filtered liquid again, preparing for a second filtration.

4. Attach the 1 mL syringe to the yellow filter membrane and perform the second filtration, transferring the liquid into a brown liquid-phase vial. Repeat to ensure complete filtration, then seal the vial.

5. The sample is prepared for HPLC analysis. (If immediate analysis is not possible, store the sample at -20°C.)

HPLC Detection

1. Use a C8 column with a mobile phase consisting of water, methanol, and acetic acid mixed in a ratio of 85:15:0.5 (v/v).

2. Detect the absorbance peak at a UV wavelength of 580 nm with a column temperature of 35°C.

Attempt to Produce 7-MX Using Coffee Grounds

Extraction of Caffeine as a Production Substrate

1. Weigh 2 g of coffee grounds from four different brands (Luckin, Starbucks, 818, Cotti) and place them into four 10 mL centrifuge tubes.

2. Add 5 mL of pH=9 Tris to each centrifuge tube and gently shake until the coffee grounds are dissolved.

3. Open the lids of the four centrifuge tubes. Place every two tubes into a 100 mL Erlenmeyer flask for stabilization.

4. Place the Erlenmeyer flasks in a 100°C water bath and heat continuously to fully dissolve the caffeine from the coffee grounds. After heating for 10 min, remove the flasks and allow them to cool to room temperature.

Production of 7-MX

1. After cooling, close the lids of the centrifuge tubes and centrifuge symmetrically at 4000 rpm, 4°C for 10 min.

2. Transfer the supernatant from the centrifuge tubes into 2 mL EP tubes and centrifuge again at 15,000 g for 10 min.

3. Aspirate 1 mL of the supernatant from the previous centrifugation into a 10 mL centrifuge tube containing 100 OD ndmB Q289A strain. Resuspend uniformly.

4. Take 50 μL of the resuspended culture into a 1.5 mL EP tube as the 0-hcontrol sample. Transfer the remaining culture into a 100 mL Erlenmeyer flask, seal with a breathable film, and ferment for 18 h. Every 2 h, take 50 μL samples for subsequent HPLC sample preparation.

Sample Preparation for 7-MX Detection

1. Add 50 μL of pH=9 Tris to each of the 10 fermentation samples in 1.5 mL EP tubes for dilution. Mix thoroughly and centrifuge at 15,000 g for 3 min.

2. After centrifugation, use a 1 mL syringe to aspirate all of the supernatant.

3. Attach a yellow filter membrane to the 1 mL syringe and filter the liquid into a brown liquid-phase vial. Repeat to ensure complete filtration, then seal the vial.

4. The sample preparation is complete and ready for HPLC analysis. (If immediate analysis is not possible, store at -20°C.)

HPLC Detection

1. Use a C8 column with a mobile phase composed of water, methanol, and acetic acid mixed in a ratio of 85:15:0.5 (v/v).

2. Detect the absorbance peak at a UV wavelength of 580 nm with a column temperature of 35°C.

![](https://static.igem.wiki/teams/5313/home/homegotop.png)

[Mutation Screening

[1. GuaB Gene Knockout - RED Homologous Recombination Method](#side1_1)
[2. Construction and Evaluation of the Sensor](#side1_2)
[3. Mutation Screening](#side1_3)](#side1)
[Exploration of optimal reaction conditions

[Substrate Concentration Gradient Experiment](#side2_1)
[Biomass Gradient Experiment](#side2_2)
[Catalysis Time Curve](#side2_3)
[Effects of Different Amino Acid Mutations on Reaction](#side2_4)](#side2)
[Practical Applications of Coffee Grounds

[1. Collection of Coffee Grounds](#side3_1)
[2. Determination the caffine titer of Coffee Grounds](#side3_2)
[3. Biosynthesis 7-MX Using Caffeine from Coffee Grounds](#side3_3)](#side3)

Results

Mutation Screening

1. *GuaB* Gene Knockout - RED Homologous Recombination Method

To construct the biosensor, a deficient strain is required, where 7-MX needs to be essential for its survival. By investigating the metabolic pathways related to xanthine, a downstream metabolite of 7-MX in Escherichia coli (E. coli )(Figure 1), it was determined that the cell’s DNA and RNA precursors are synthesized through two pathways. The primary pathway involves the *guaB* gene, while the secondary pathway utilizes xanthine as a substrate and is mediated by the gpt gene. Therefore, knockout of the *guaB* gene to disrupt the primary pathway will activate the secondary pathway and create the desired deficient strain.

![](https://static.igem.wiki/teams/5313/experiment/result/result1.png)

Figure 1 Xanthine metabolism chart

To achieve this, the RED homologous recombination method was used to knock out the *guaB* gene and replace it with a kanamycin (Kana) resistance gene for subsequent experiments.Detailed experimental procedures can be found on our experiment page.

First, we constructed the BW/pKD46 electrocompetent strain and utilized PCR to amplify a homologous recombination fragment containing a resistance marker (Kana) to delete the *guaB* gene, resulting in the *BW-ΔguaB* strain. Meanwhile, a targeting fragment was prepared by amplifying approximate 500 bp sequences flanking the *guaB* gene through PCR.

Next, the assembled fragment was electroporated into the induced *BW-ΔguaB* electrocompetent cells to achieve gene knockout.The resulting knockout strains were then subjected to colony PCR screening , with original strains used as negative controls. The results are as follows (Figure 2).

![](https://static.igem.wiki/teams/5313/experiment/result/result2.png)

Figure 2 Colony PCR to screen for the knockout clones using the upper and lower homology arms as primers

Here, the length of the Kana resistance gene used for replacement is 1600 bp, while the length of the *guaB* gene to be knocked out is 2000 bp. Preliminary evidence indicated successful knockout, followed by sequence analysis of the fragment and testing under selective culture conditions, with results consistent with expectations.

Summary：These results showed that the knockout process of *guaB* gene was smooth and the strain performed well under selective culture conditions, which laid a foundation for subsequent experiments.

2. Construction and Evaluation of the Sensor

Successful knockout strains were isolated and subjected to single-colony expansion to produce competent *BW25113-ΔguaB* cells, which are now xanthine-dependent strains.Subsequently,based on previous literature[1] the ndm gene family responsible for methylxanthine degradation was utilized to design the pYB1s-ndmDCE plasmid (pDCE) (Figure 3).

This plasmid carrys a system that degrades 7-MX into xanthine.

![](https://static.igem.wiki/teams/5313/experiment/result/result3.png)

Figure 3 Plasmid pYB1s-ndmDCE

Using the Golden Gate method, the PCR-amplified ndmC, ndmD, and ndmE coding sequences were ligated into the pYB1s vector. Subsequently, colony PCR was performed using ndmC-F and ndmE-R primers to screen for the successful insertion of these genes. The results are shown in Figure 4.

![](https://static.igem.wiki/teams/5313/experiment/result/result4.png)

Figure 4 Results of colony PCR screening validation

The lengths of the PCR products were as expected, indicating the pDCE was successfully constructed.

The plasmid was then transformed into competent *BW25113-ΔguaB* cells, which can allow these cells to utilize 7-MX for DNA and RNA synthesis, and thus make 7-MX essential for their survival. Consequently, this process converted the strain into a sensor to detect 7-MX levels.

Thereby preliminarily constructing a biosensor. We then proceeded to test the functionality of the sensor, focusing on two key aspects: substrate specificity and substrate concentration sensitivity.

We first examined the substrate specificity by dissolving seven types of methylxanthines and xanthines in M9 medium. During the induction of the bacterial cells, to enhance bacterial proliferation, we used ZY medium supplemented with double the amount of yeast extract and LB medium. In the subsequent steps of transferring the cells, the induced bacterial cells were washed with water to remove the yeast extract from the ZY medium, which contains xanthine, and were then washed and resuspended in M9 medium. Finally, various methylxanthine compounds were individually added to a 96-well plate, followed by the addition of 200 μL of the bacterial suspension to each well. The growth curves were monitored using a microplate reader. The results are shown in Figure 5.

![](https://static.igem.wiki/teams/5313/experiment/result/result5.png)

Figure 5 OD value of the strain at 36 h

The growth curve results obtained from the microplate reader indicate that the *BW25113-ΔguaB* strain transformed with the pDCE exhibited the expected dependence on 7-MX and xanthine. Specifically, when the strain was cultured in medium containing 7-MX, its growth rate increased significantly, suggesting that 7-MX serves as an essential compound for this strain. Furthermore, by testing the effects of other methylxanthine compounds, we found that the biosensor exhibited a high specificity for 7-MX, as other methylxanthines did not show the same growth-promoting effect.

Thus, the substrate specificity of the sensor has been successfully confirmed.

Next, we tested the concentration gradient of 7-MX. Using the same method as before, we dissolved 7-MX in the M9 medium at concentrations ranging from 0.1 to 0.5 mM/L. Following the same procedure, we used a microplate reader to monitor the growth curves. The results are shown below (Figure 7).

![](https://static.igem.wiki/teams/5313/experiment/result/result6.jpg)

Figure 7 Growth curves of experimental bacteria in different concentrations of 7-MX in an enzyme labeling apparatus over a period of 36 h

The experimental results demonstrate that the biosensor exhibits good sensitivity to changes in 7-methylxanthine (7-MX) concentration. As the 7-MX concentration increases, the growth rate of the cells shows a clear dependence. Under low 7-MX concentration (0.1 mM/L), the cell growth rate is relatively slow, but as the concentration rises (to 0.3 mM/L and 0.5 mM/L), cell growth accelerates significantly, indicating that the strain's demand for and response to 7-MX strengthens with increasing concentration.

Summary: These experiments confirm that the biosensor exhibits the expected substrate specificity and concentration sensitivity. 7-MX can indeed be accurately detected by the sensor and serves as an essential factor for cell growth, providing a solid foundation for further experimental applications.

3. Mutation Screening

3.1 First Round of Screening

We conducted site-directed mutagenesis on five key predicted sites for both NdmA and NdmB, as reported in the literature. Corresponding degenerate primers were designed to perform site-directed random mutagenesis, thereby constructing mutant libraries for these two genes.

For the first round of screening and validation (Figure 7), the products of the directed mutations underwent DNA purification and DpnI digestion to minimize the background caused by the original template plasmid. The mutant fragments were then ligated to the vector and transformed into DH5α competent cells. After overnight cultivation, the monoclonal colonies on the plates were washed off and pooled using sterile water. Plasmids were then extracted from the mixture and transformed into BW25113 competent cells. The transformation products were amplified by shaking incubation, and single colonies were isolated using a dilution plating method.After incubating the plates for a period of time, single colonies were picked and inoculated into deep-well plates for further cultivation. Caffeine substrate was added to the deep-well plates to induce expression and perform whole-cell catalysis. Subsequently, 200 μL of the transformation solution and 10 μL of the induced biosensor strain were transferred into a 96-well plate. A microplate reader was then used to detect whether the mutated strain could produce 7-MX, allowing the biosensor strain to grow normally, thus validating the function of the mutations.

![](https://static.igem.wiki/teams/5313/experiment/result/result7.png)

Figure 8 The overall process of mutation screening

Subsequent screening of the mutated strains revealed that the NdmA mutants did not meet expectations, although a few NdmB mutants showed promising results (Figure 9). Consequently, we decided to continue further mutational screening of the *ndmB* gene to obtain strains more aligned with the desired outcomes (Figure 8).

![](https://static.igem.wiki/teams/5313/engineering-new/engineering-fig-21-ndmas.png)
![](https://static.igem.wiki/teams/5313/engineering-new/engineering-fig-21-ndmbs.png)

Figure 9 Growth of mutant bacteria detected by microplate reader

3.2 Second Round of Screening

In the second round of mutations, to clarify the key active sites, the five critical pocket residues in NdmB were divided into two groups based on their proximity. One group consisted of two closely located sites (Gln289 and Leu293), while the other group included three more dispersed sites (Trp256, Cys267, and Met271). Site-directed mutagenesis was performed on both groups to obtain mutants from random combinations of mutations at these sites (Figure 10).

![](https://static.igem.wiki/teams/5313/experiment/result/result10.png)

Figure 10 The five key pocket residues in NdmB

The screened mutant strains underwent a second round of validation and screening.

After recombining the mutant plasmids, we first transformed them into DH5α. Single colonies were grown, and the plasmids were extracted and subsequently transformed into BW25113 and plated. Single colonies were then picked and cultured in deep-well plates with 800 μL of ZY medium containing Ara, IPTG and S50, and induced at 25°C for 18 h. The deep-well plates were centrifuged at 4200 rpm for 10 min, and the supernatant was discarded. Then, 400 μL of 1× M9 medium supplemented with 0.5 mM caffeine (representing a 2x concentration of the cell mass) was added. Whole-cell catalysis was performed at 25°C for 18 h, followed by centrifugation. 200 μL of the supernatant was transferred into a 96-well plate, and 10 μL of a supplement solution (containing ZYM5052, calcium chloride, magnesium chloride, trace elements, inducers, and antibiotics) was added. Another 10 μL of pre-induced pDCE (using double the amount of yeast extract) was added (as pDCE may grow slowly, the 96-well plate was sealed in a plastic bag and stored at 4°C until pDCE induction was complete).

The OD values of each strain were measured using a microplate reader (Figure 11).

![](https://static.igem.wiki/teams/5313/engineering-new/engineering-fig-23.png)

Figure 11 OD value for 24 h of growth

Mutant strains with good growth (OD ≥ 1.5) were selected for HPLC analysis to determine whether any products were produced (Figure 12).

![](https://static.igem.wiki/teams/5313/experiment/result/result15.png)

Figure 12 The well-growing mutant strains were detected by HPLC

The strains producing only 7-MX with the highest yield were identified as target mutant strains. A selected highest 7-MX-producing strain was then sequenced, and the sequencing results revealed that the NdmB protein had only one single mutation at the position 289, where glutamine was substituted by alanine, while other minor mutations were synonymous. This suggests that the 289 position is a critical for the catalytic activity of NdmB. At this stage, we cannot exclude the possibility that mutating this position to other amino acids could yield strains with higher 7-MX production yields.

To explore this hypothesis, a site-directed saturation mutagenesis was performed at position 289, followed by the same cultivation and screening process as described above. Ultimately, the results showed that the strain with the highest yield remained the one with the Q289A (glutamine to alanine) mutation, confirming its optimal performance.

Exploration of optimal reaction conditions

Based on the screening results, the NdmBQ289A strain was selected for subsequent exploration of optimal whole cell catalysis conditions.(Figure 13)

![](https://static.igem.wiki/teams/5313/experiment/result/result1666.jpg)

Figure 13 The overall process of exploring optimal reaction conditions

Schematic diagram of the experimental procedure that is primarily involved conducting gradient experiments on both whole cell catalysis biomass and substrate concentration to analyze the relationship between biomass, substrate concentration and yield.

Substrate Concentration Gradient Experiment

Initially, a gradient experiment to test substrate concentration was performed. It was anticipated that higher substrate concentrations would lead to better 7-MX yield. Using a biomass of 50 OD, caffeine at a gradient of concentrations was used as the substrate for the whole-cell catalysis, and the product yields were measured by HPLC. The results are shown in Figure 14.

![](https://static.igem.wiki/teams/5313/experiment/result/result17.png)

Figure 14 Bar chart of the effects of different substrate concentrations on 7-MX production with experimental strains under 50 OD conditions

Based on the results, we concluded that, under the 50 OD biomass condition, a substrate concentration of 4 mM allowed complete conversion of caffeine, with a 7-MX yield of approximate 3.5 mM. However, there was an accumulation of the intermediate product PX. When the substrate concentration exceeded 4 mM, the results deviated from the expected outcome. Although caffeine was almost entirely converted, the yield of 7-MX did not increase significantly; instead, the accumulation of PX continued to rise.

Based on these data, the maximum yield of 7-MX under 50 OD biomass conditions is limited to 4 mM, suggesting that biomass might be the primary limiting factor to improve 7-MX production.

Biomass Gradient Experiment

Considering that a 10 mM substrate concentration can still be significantly converted and that the accumulation of intermediate products is high, we conducted the subsequent experiment using a 10 mM substrate to explore whether biomass limitations affect 7-MX production. The results are shown in Figure 15.

![](https://static.igem.wiki/teams/5313/experiment/result/result18.png)

Figure 15 The bar chart of the effects of different OD value on 7-MX production with experimental strains under 10mM caffeine

The HPLC results fully met the experimental expectations. Increasing the biomass directly enhanced the molar ratio of 7-MX in the final system. Ultimately, under conditions of 100 OD biomass and 10 mM substrate concentration, a conversion rate of 90% from caffeine to 7-MX was achieved. As biomass increased, the substrate conversion rate was noticeably improved. However, if the biomass is increased before the complete conversion of caffeine, there is a significant accumulation of the intermediate product PX. It is only after the complete consumption of caffeine that increasing biomass helps reduce the accumulation of PX.

Catalysis Time Curve

Given the substantial accumulation of the intermediate product PX, we hypothesized that the conversion of PX to 7-MX is the rate-limiting step. To validate this hypothesis, timed sampling during the whole-cell catalysis process was conducted to analyze the changes in the concentrations of different substances over time, and corresponding catalysis time curves were plotted (Figure 16).

![](https://static.igem.wiki/teams/5313/experiment/result/result19.png)

Figure 16 Time curve of the production of 7-MX with experimental strains at 10mM caffeine, 50OD

Under conditions of 100 OD biomass and 10 mM caffeine substrate, the whole-cell catalysis process for 18 h achieved complete conversion of caffeine within 4 h. Subsequently, the intermediate product PX was slowly converted into the product 7-MX. However, the conversion rate significantly decreased over time, possibly due to the rapid inactivation of the enzyme. Due to time constraints, further experiments to verify changes in enzyme activity during this process were not conducted. Investigating this in more detail could potentially optimize the procedure to further increase the yield of 7-MX.

Effects of Different Amino Acid Mutations on Reaction

In the sensor screening phase, three strains were identified with mutations at the position 289, where the amino acids were mutated to Thr, Ser, and Ala, respectively. After optimizing the whole cell catalysis conditions, the yields of these three strains were re-evaluated (Figure 17).

![](https://static.igem.wiki/teams/5313/parts/tubian-result-17.png)

Figure 17 Different mutated strains were detected by HPLC

Using the same conditions of 100 OD biomass and 10 mM caffeine substrate, the results still indicate that the NdmBQ289A strain achieves the highest yield of 7-MX. Interestingly, an additional observation was made: the original NdmB enzyme only removes the methyl group at the N3 position, converting caffeine into PX (the intermediate product observed in the experiment). However, with mutations leading to smaller R groups, while the enzyme's ability to remove the N3 methyl group remains effective, there is an increasing capability to remove the N1 methyl group. This suggests that the smaller R group at position 289 allows the substrate to enter the active site from different orientations, resulting in different outcomes. This finding raises a hypothesis that NdmB and NdmA may have originally been the same enzyme, which through a long evolutionary process, gradually diverged in their functions.

Practical Applications of Coffee Grounds

1. Collection of Coffee Grounds

Initially, coffee grounds were collected from various coffee shops, such as Starbucks, Luckin Coffee, Kudi, and 818(Figure 16). During the collection process, inquiries were made regarding the disposal methods for coffee grounds. As anticipated, the majority of coffee shops opted to discard the coffee grounds directly.

![](https://static.igem.wiki/teams/5313/experiment/result/result21.jpg)
![](https://static.igem.wiki/teams/5313/experiment/result/result22.jpg)

Figure 18 Team members go to a coffee shop to get coffee grounds

![](https://static.igem.wiki/teams/5313/experiment/result/result23.png)

Figure 19 Different brands of coffee grounds obtained

Upon observing the collected coffee grounds, it was noted that the grounds from Starbucks and Luckin Coffee were initially processed into clumps, while those from other sources remained unprocessed. The coffee grounds were then spread out in foam trays and placed in an oven set at 60°C to dry for 48 h, resulting in dehydrated coffee grounds (Figure 19).

2. Determination the caffine titer of Coffee Grounds

Base on previous literature, we attempted to extract caffeine from the coffee grounds. Gradient dilution extraction was first performed on the coffee grounds from Luckin Coffee and Starbucks to extract caffeine(Figure 20).

![](https://static.igem.wiki/teams/5313/experiment/result/result24.png)

![](https://static.igem.wiki/teams/5313/experiment/result/result25.png)
![](https://static.igem.wiki/teams/5313/experiment/result/result26.png)

Figure 20 Extract caffeine from Luckin and Starbucks coffee grounds

However, according to the liquid chromatography data, it was concluded that the caffeine content in each gram of coffee grounds is between 90-100%. This significantly differs from the theoretical values initially reviewed, which should be in the range of 0.5-1.5% [2](Figure 21).

![](https://static.igem.wiki/teams/5313/experiment/result/result27.png)
![](https://static.igem.wiki/teams/5313/experiment/result/result28.png)

Figure 21 Bar chart of the concentration of caffeine extracted from Luckin and Starbucks coffee grounds

Since the results did not meet expectations, an analysis of the process was conducted. We predicted that during the ultrasonic extraction of caffeine, the ultrasonic waves might have caused partial decomposition of substances in the coffee grounds (such as lipids and proteins), leading to inaccurate measurement. Additionally, insufficient dilution might have resulted in incomplete extraction. Therefore, we decided to increase the dilution factor while simultaneously use heating method to improve caffeine extraction.

For each brand of coffee grounds, 5 grams were weighed and dissolved in 100 mL of water. The mixture was then heated in a microwave until boiling, which was maintained for five times. After samples cooling down, the solution for each sample was filtered using filter paper. The filtered liquid was then analyzed by HPLC. The filtered solution was passed through the 0.22μm membrane one more time to prevent the presence of any solid residues to clog the HPLC chromatographic column (Figure 22).

![](https://static.igem.wiki/teams/5313/experiment/result/result2999.jpg)

Figure 22 The overall process of extracting caffeine from coffee grounds

The final results indicate that the coffee grounds content of the four brands falls within the range of 0.5–1.5%. Furthermore, using the extraction method referenced in the literature, our results, after accounting for the loss of caffeine, are largely consistent with the reported value of 2 milligrams of caffeine per gram of coffee grounds (Figure 23).

![](https://static.igem.wiki/teams/5313/experiment/result/result30.png)

Figure 23 Bar chart of the concentration of caffeine extracted from 4 different brands

3. Biosynthesis 7-MX Using Caffeine from Coffee Grounds

After measuring the caffeine content in the coffee grounds, we decided to directly use the coffee grounds to prepare a whole-cell catalysis reaction mixture. Subsequently, the optimized strains were employed to utilize this reaction mixture derived from the coffee grounds for the production of 7-MX.

![](https://static.igem.wiki/teams/5313/experiment/result/result31.png)

Figure 24 The whole cell catalyzed conversion solution was made from 4 different brands of coffee grounds

The extraction was carried out using 10 mL centrifuge tubes. Each brand of coffee grounds was weighed at 2 grams and dissolved in 5 mL of Tris buffer (Figure 24). Due to the small size of the container and the risk of boiling over, a water bath heating method was used(Figure 25a and b) The centrifuge tubes were placed in boiling water and heated for 10 min. After cooling, the mixture was centrifuged at 4000 rpm for 10 min. The supernatant was then transferred to a 2 mL Eppendorf tube and subjected to a second round of centrifugation at 15,000 g for 10 min (Figure 25c).

![](https://static.igem.wiki/teams/5313/experiment/result/result32.jpg)
![](https://static.igem.wiki/teams/5313/experiment/result/result33.jpg)

![](https://static.igem.wiki/teams/5313/experiment/result/result34.png)
![](https://static.igem.wiki/teams/5313/experiment/result/result35.png)

Figure 25.a Water bath heating fixture device .b Integrated device for water bath heating method.c Procedures before 18 h fermentation.d Mix the NdmBQ289A strain with transformation liquid containing caffeine.

Then, whole-cell catalysis was performed using the induced NdmBQ289A strain. One milliliter of the transformation liquid was added to 100 mL of the culture medium with 100 OD of the bacteria in a flask.(Figure 25d)The whole-cell catalysis was conducted under the optimized conditions for 18 h. Starting from 0 h, a 50 μL aliquot of samples was collected every 2 h, diluted by a factor of two, and prepared for HPLC analysis to generate the whole-cell catalysis time curve (Figure 26).

![](https://static.igem.wiki/teams/5313/experiment/result/result36.png)

Figure 26 The fermentation curves with 100 OD NdmBQ289A over 18 h

According to the HPLC results, the outcomes fully align with the experimental expectations, showing that, from 0 h with no 7-MX present, caffeine was almost entirely converted into 7-MX and PX by the end. However, the conversion rate of caffeine to 7-MX was lower than that using the pure caffeine. This suggests that some components in the coffee grounds may interfere with the enzyme's activity.

[1] Kim, J. H., Kim, B. H., Brooks, S., Kang, S. Y., Summers, R. M., & Song, H. K. (2019). Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex. Journal of molecular biology, 431(19), 3647–3661. <https://doi.org/10.1016/j.jmb.2019.08.004>

[2] Vandeponseele, Alexandre, Micheline Draye, Christine Piot, and Gregory Chatel (2021). Study of Influential Parameters of the Caffeine Extraction from Spent Coffee Grounds: From Brewing Coffee Method to the Waste Treatment Conditions. Clean Technologies 3, no. 2: 335-350.<https://doi.org/10.3390/cleantechnol3020019>