iGEM 2024 — Team njtech-china-a

![](https://static.igem.wiki/teams/5366/home/home-picture/band.png)
[NJTech-China-A](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27home%27%29%20%7D%7D)

* [Project](#option1)

  [Description](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27description%27%29%20%7D%7D)
  [Contribution](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27contribution%27%29%20%7D%7D)
  [Protocol](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27protocol%27%29%20%7D%7D)
  [Engineering](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27engineering%27%29%20%7D%7D)
  [Experiments](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27experiments%27%29%20%7D%7D)
  [Notebook](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27notebook%27%29%20%7D%7D)
  [Results](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27results%27%29%20%7D%7D)
* [Part](#option2)

  [Part](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27part%27%29%20%7D%7D)
* [Model](#option3)

  [Model](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27model%27%29%20%7D%7D)
* [Team](#option4)

  [Members](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27members%27%29%20%7D%7D)
  [Attribution](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27attributions%27%29%20%7D%7D)
* [Human Practices](#option5)

  [Human Practice](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27human-practices%27%29%20%7D%7D)
  [Education](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27education%27%29%20%7D%7D)
  [Communication&Collaboration](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27communication%27%29%20%7D%7D)

# Notebook

* [New enzymatic mining of tagatose-4- epimerase](#section1)
* [The soluble expression of the AJC7](#section2)
* [Mutation of AJC7](#section3)
* [Study of the biosensors](#section4)

New enzymatic mining of tagatose-4- epimerase

2024.5.20

The amino acid sequence UxaE, derived from *Thermotoga* *petrophila* RKU-1, was utilized as a template to conduct a search in the non-redundant protein sequence database of the National Center for Biotechnology Information (NCBI) using the BLASTp function. This search selected 100 sequences with the highest sequence identity. A phylogenetic tree was constructed using MEGA software.

2024.5.21-5.23

Eighty sequences with a similarity threshold below 70% were selected from the 100 sequences screened by BLASTp, and these sequences were structurally modeled using AlphaFold2.

2024.5.24-5.28

Molecular docking of the selected sequences and the binding free energy between them and fructose were calculated. Four proteins of unknown function with tagatose-4-epimerase activity were selected and designated as MBC, AJC7, TET, and HDM.

2024.6.4

The resultant plasmids pET-28a(+)–AJC7, pET-28a(+)–HDM, pET-28a(+)–MBC, pET-28a(+)–TET, and pET-28a(+)–UxaE were transferred into *E. coli* BL21 (DE3) and incubated at 37°C for 14 hours.

2024.6.5

Individual colonies were picked from those grown after transformation and subjected to colony PCR. The correct single colony identified by colony PCR was transferred into LB containing kanamycin for activation.

2024.6.6

The activated bacterial solution was transferred into 50 mL of LB containing kanamycin, and at an OD600 of 0.6, IPTG was added to a final concentration of 0.5 mM, then incubated at 20°C for 16 hours.

2024.6.7

Proteins were extracted and purified for tagatose production. The samples were analyzed using high-performance liquid chromatography (HPLC).

2024.6.9

Based on the tagatose titer analyzed by HPLC, the best wild-type enzyme selected was AJC7.

The soluble expression of the AJC7

2024.6.14

To build the plasmid with the fusion tag, the tag was designed using SnapGene to connect to the plasmid vector containing the target gene. The corresponding primers were designed and sent to the company for synthesis to confirm the template plasmid.

2024.6.17

1.The plasmid containing the AJC7 vector and fusion tag fragments was amplified by PCR to generate the AJC7 vector and the corresponding pro-melting tag fragments. The amplified fragments were verified by nucleic acid gel electrophoresis. After confirming their correctness, the gel was processed for fragment recovery. The concentrations of the recovered fragments and carriers were measured and stored at -20°C for subsequent use.

2.The fragments and vectors were cloned in a single step, and the resulting products were transformed via heat shock. Following the heat shock transformation, a certain volume of bacterial solution was spread on LB plates containing the appropriate antibiotic for preliminary screening. The plates were inverted and incubated for 12 hours at 37°C.

2024.6.18

Five to seven colonies were selected from each plate and verified by colony PCR. The confirmed colonies were cultured in 5 mL centrifuge tubes for 12 hours for subsequent testing.

2024.6.20

The correct strains will be sequenced, maintained, and expanded in culture. When the optical density (OD600) reaches 0.6, 5 µL of IPTG will be added, and the culture will be incubated at 20°C for 16 hours.

2024.6.21-6.28

Post-induction, bacteria were collected, sonicated, and the supernatant containing cell lysates was obtained by centrifugation. Protein expression was analyzed using SDS polyacrylamide gel electrophoresis, and the optical density of the protein bands was assessed. For re-induction of recombinant plasmids showing incorrect protein bands, the protein was extracted.

2024.6.29

The molecular chaperone plasmid was transformed alongside the recombinant plasmid into competent cells and cultured on plates containing chloramphenicol (Cm) and kanamycin (Kan) for 12 hours.

2024.6.30-7.2

Single colonies confirmed by colony PCR were activated in LB media with Cm and Kan. Colony PCR results were verified for correct re-transformation.

2024.7.3

The activated bacterial cultures were transferred into 50 mL LB media containing the appropriate antibiotics and chaperone inducers:

|  |  |
| --- | --- |
| Molecular chaperones | Mutagen |
| pG-KJE8 | L-Arabinose、Tetracyclin |
| pGro7、pKJE7、pTf16 | L-Arabinose |
| pG-Tf2 | Tetracyclin |

When the OD600 reached 0.6, IPTG was added to a final concentration of 0.5 mM and the cultures were induced at 20°C for 16 hours.

|  |  |  |  |
| --- | --- | --- | --- |
| Cycle steps | Temperature | Time | Cycles |
| Predegeneration | 98°C | 30 sec |  |
| Transgender | 98°C | 10 sec |  |
| Annealing | Tm | 5 sec | 28-35cycles |
| Extension | 72°C | 5 ~ 10 sec/kb |  |
| Completely Stretch | 72°C | 1 min |  |

2024.7.4

Bacterial cells were harvested, and protein was extracted. Protein expression was assessed by SDS polyacrylamide gel electrophoresis, and the optical density of the protein bands was analyzed.

2024.7.5-7.12

Inducer and temperature gradients were established to investigate the effects of varying temperatures and inducer concentrations on protein solubility. Protein expression was again determined by SDS polyacrylamide gel electrophoresis, with analysis of the optical density of the protein bands.

2024.7.12-2024.7.20

The effect of auto-induced medium was assessed by establishing orthogonal combinations of different carbon source media. Protein expression was analyzed using SDS polyacrylamide gel electrophoresis, and the optical density of the protein bands was examined.

Mutation of AJC7

The corresponding bands were quickly excised under UV light for gel recovery using the Novizen Product Purification Kit.

2024.7.7

Primer design:The upstream and downstream primers, including AJC7-S125D-F, AJC7-S125D-R, AJC7-T181A-F,AJC7-T181A-R, AJC7-H342L-F, AJC7-H342L-R，AJC7-I129T-R, AJC7-I129T-F, AJC7-L140P-F, and AJC7-L140P-R, were designed for synthesis.

2024.7.9

1.Point mutations were introduced into the plasmid through PCR, and the template plasmid was eliminated using DpnI.

2.PCR product recovery: The accuracy of the amplified products was verified via agarose gel electrophoresis, and the desired fragments were recovered from the agarose gel.

2024.7.10

The constructed plasmids were transformed into competent *E. coli* BL21 cells, and the bacterial solution was spread on LB agar plates containing kanamycin, then incubated overnight at 37°C.

2024.7.11

Well-growing monoclonal cells were selected for expanded culture and induced expression. The cell cultures were subsequently sent to a professional testing facility for sequencing verification.

2024.7.13

When the correctly sequenced bacterial culture reached an OD600 of 0.6, IPTG was added to a final concentration of 0.5 mM. The culture was then incubated at 20°C. for 16 hours to induce protein expression.

2024.7.14

Protein was extracted and purified from the induced bacterial culture. The pure enzyme solution was then reacted with 10 g/L fructose in a 1 mM Ni2+ Tris-HCl buffer at pH 8, incubated at 70°C. for 5 hours. The reaction products were analyzed using high-performance liquid chromatography (HPLC) to assess the outcomes of the enzymatic reaction.

2024.7.15

The HPLC results indicated that the AJC7-S125D mutant strain exhibited the highest enzyme activity among the tested samples.

2024.7.16-7.23

The experiments were repeated, and consistent with previous findings, the HPLC results again demonstrated that the AJC7-S125D mutant strain exhibited the best enzyme activity. This reinforces the potential of this mutant for further studies or applications.

2024.7.24-7.30

Two-point mutants, specifically S125D/T181A, S125D/H342L, S125D/I129T, and S125D/L140P, were engineered for further study.

2024.7.31-8.7

Transformation, induction, protein extraction, purification, and HPLC analysis were conducted on the two-point mutants. The results indicated that S125D/T181A was the optimal mutant among those tested.

2024.8.8-8.22

Three-point mutants, specifically S125D/T181A/I129T, S125D/T181A/L140P, and S125D/T181A/H342L, were constructed. Transformation, induction, protein extraction, purification, and HPLC analysis were performed on these mutants. The results revealed that S125D/T181A/I129T was the optimal three-point mutant.

2024.8.22-2024.9.8

The four-point mutant S125D/T181A/I129T/L140P and the five-pointmutant S125D/T181A/I129T/L140P/H342L were constructed. Transformation, induction, protein extraction, and purification of the four-point mutant were performed,followed by reaction and HPLC detectionof the products.

2024.9.8-9.14

The data analysis revealed that the S125D/T181A/I129T triple mutant exhibited the best performance among all mutants. Consequently, the enzymatic properties and kinetic parameters of the S125D/T181A/I129T triple mutant were assessed. The process included activation, transformation, induction, and protein extraction of the optimal mutant S125D/T181A/I129T. A pure enzyme solution of the S125D/T181A/I129T triple mutant was obtained, and SDS-PAGE was performed to verify the protein and determine enzyme concentration.

2024.9.14-9.18

Enzymatic properties and kinetic parameters were assessed for the purified enzyme solution, as detailed in the [Experimental] section. Product yield was evaluated using high-performance liquid chromatography.

Study of the biosensors

2024.7.15

Primer design: The upstream primers and downstream primers PetDuet-1-pd-For and pETDuet-1-tagR-P-1-tagR-sfGFP-FOR, T7T, were designed by plasmid mapping.Send it to the company for the synthesis.

2024.7.17

1.The fragments from the pETDuet-1-gene cluster and pETDuet-1-tagR-P-RS-sfGFP plasmids were amplified via PCR, followed by verification through nucleic acid gel electrophoresis. After confirming correctness, glue recovery was performed, and the concentrations of the recovered fragments and vectors were measured and stored at -20°C for future use.

2.The fragments and vectors were cloned in one step, and the connected products were transformed using a heat shock method. Following transformation, a portion of the bacterial liquid was spread on LB plates containing the appropriate antibiotic for preliminary screening. The plates were inverted and incubated for 12 hours at 37°C.

2024.7.18

Five to seven spots were selected from each plate and verified using colony PCR. The correctly verified colonies were then cultured in 5 mL centrifuge tubes for 12 hours. Afterward, the cell cultures were sent to a professional testing facility for sequencing confirmation.

2024.7.20

1.The correct bacterial solution will be subjected to sequencing to confirm the presence of the desired insert.

2.The correctly sequenced pETDuet-1-tagR-P-RS-sfGFP plasmid and the pETDuet-1-gene cluster plasmid were simultaneously transformed into BL21 (DE3) competent cells, which were then grown overnight at 37℃.

2024.7.21

After selecting well-growing monoclonal cells for expanded culture and reaching an OD600 of 0.6, expression was induced with 30 mM IPTG for 5 hours. However, no green fluorescence was detected, indicating a potential issue with expression or protein folding.

2024.7.22

To address the lack of green fluorescence, review literature on the following: optimal induction conditions (e.g., IPTG concentration and timing), temperature adjustments during induction, and plasmid compatibility. Consider testing different host strains or adding specific chaperones to enhance protein folding. Adjust the experimental scheme based on these findings for improved expression.

2024.7.23

1.The strain containing the correctly sequenced pETDuet-1-tagR-P-RS-sfGFP plasmid was made competent for transformation.

2.The sequenced pETDuet-1-gene cluster plasmid was then introduced into the competent cells harboring pETDuet-1-tagR-P-RS-sfGFP and cultured overnight at 37°C.

2024.7.24

No cells grew on the plates after the overnight culture.

2024.7.25

The transformation was repeated and grown overnight at 37℃.

2024.7.26

Cells exhibiting optimal growth were selected for expanded culture until an OD600 of 0.6 was reached. Induction was performed at a final IPTG concentration of 30 mM, followed by expression at 20°C, 30°C, and 37°C for 5 hours. However, no green fluorescence was detected.

2024.7.27

1.To identify the strain containing the correctly sequenced pETDuet-1 gene cluster plasmid.

2.The correctly sequenced pETDuet-1-tagR-P-RS-sfGFP plasmid was introduced into competent cells carrying the pETDuet-1 gene cluster and cultured overnight.

2024.7.28

Cells with optimal growth were selected for expanded culture until an OD600 of 0.6. Induction was carried out at a final IPTG concentration of 30 mM, followed by expression at 20°C, 30°C, and 37°C for 5 hours; however, no green fluorescence was detected.

2024.8.3

The upstream primers for the pACYCDuet-1-tagR-P-RS-sfGFP1 plasmid, namely ECOLINRS18700-GFP-F, Amp/Cmr-F, ECOLINRS18700-GFP-R, and Amp/Cmr-R, were synthesized by the company.

2024.8.5

1.The fragment and vector of the pACYCDuet-1-tagR-P-RS-sfGFP1 plasmid were amplified via PCR, followed by verification through nucleic acid gel electrophoresis. After confirming the accuracy of the amplified fragments, gel recovery was performed. The concentrations of the recovered fragments and vectors were measured, and they were stored at -20°C for future use.

2.The fragments and vectors were cloned in a single step, and the ligation products were transformed into competent cells using a heat shock method. After the transformation, a portion of the bacterial solution was spread on LB plates containing the appropriate antibiotic for preliminary screening. The plates were then inverted and incubated overnight at 37°C.

2024.8.6

Well-growing colonies on the plates were selected for colony PCR validation. The colonies that passed verification were then cultured in 5 mL centrifuge tubes for 12 hours. After the incubation, the cell cultures were sent to a professional testing facility for sequencing confirmation to ensure the correctness of the cloned fragments.

2024.8.8

1.Prepare the Nissle 1917 (EcN) competent cells for transformation.

2.The correctly sequenced plasmids were then introduced into the competent Nissle 1917 (EcN) cells. Following the transformation, the cells were incubated overnight at 37°C to allow for expression and replication of the plasmid.

2024.8.9

Well-growing cells were selected for expanded culture, reaching an OD600 of 0.6. Induction was carried out at a final concentration of 30 mM, with expression conducted at 20°C, 30°C, and 37°C for 5 hours. However, no green fluorescence was detected, indicating a potential issue with expression or protein folding.

2024.8.10-2024.8.12

Repeat the 8.8-8.9 experiments, and still no green fluorescence was detected.

2024.8.13

Primers were designed for the pACYCDuet-1 plasmid, including theupstream primer for P trc-gf RS and downstream primer P-RS-gfp-Rev.Additional primers such as Term-For, TagR-Rev, Ptac-For, ACYCDuetUP1 Primer,and T7 Terminator Primer were also synthesizedby a professional company.This will facilitate subsequent cloning and expression experiments.

2024.8.15

1.The fragments and vector for the designed pACYCDuet-1 P trc-tagR-P-RS-sfGFP plasmid were successfully amplified by PCR and verified through nucleic acid gel electrophoresis. After confirming the correct sizes, the fragments were subjected to gel recovery, with concentrations measured and stored at -20°C for future use.

2.The fragments and vector were cloned in one step and the ligation products were transformed into competent cells via heat shock. After transformation, a portion of the bacterial solution was plated on LB agar containing the appropriate antibiotic for preliminary screening. The plates were inverted and incubated overnight at 37°C.

2024.8.16

Well-growing colonies from the plates were selected for colony PCR validation. The colonies that tested positive for the correct insert were picked and cultured in 5 mL centrifuge tubes for 12 hours. After this incubation period, the cell cultures were sent to a professional testing facility for sequencing confirmation to verify the accuracy of the cloning and ensure the integrity of the construct.

2024.8.18

The correctly sequenced plasmids were transformed into Nissle 1917 (EcN) competent cells and incubated overnight at 37°C. This step allows for the expression of the plasmid in the EcN strain, preparing for further experiments or analysis.

2024.8.19

Well-growing cells were selected for expanded culture until they reached an OD600 of 0.6. Induction was carried out with a final concentration of 30 mM for protein expression at 20℃, 30℃ and 37℃ for 5 hours.

2024.8.19-2024.8.23

Replacement of the previous LB medium with M9 medium, and the experimental content of 7.23-7.28 and 8.8-8.9 were repeated, and still no green fluorescence was detected.

2024.8.23

Primer design: The upstream primer of the pCDFDuet-P1-sfGFP1 and P-tagR-For, pcdfduet-P1-tagR-f R-GF P-REV were sent to the company for synthesis.

2024.8.25

1.The fragments and vector for the designed pCDFDuet-P1-sfGFP1 plasmid were amplified using PCR, and the amplified products were verified through nucleic acid gel electrophoresis. After confirming the correct sizes, gel recovery was performed on the fragments and vector. The concentrations of the recovered fragments and vector were measured and subsequently stored at -20°C for later use.

2.The fragments and vector were cloned in a single-step reaction, and the ligation products were transformed into competent cells via heat shock. After the heat shock transformation, an aliquot of the bacterial culture was plated on LB agar containing the appropriate antibiotic for preliminary screening. The plates were inverted and incubated overnight at 37°C to allow for colony growth.

2024.8.26

Well-growing colonies were selected from the plates for colony PCR validation. The colonies confirmed to be correct were cultured in 5 mL centrifuge tubes for 12 hours. Following this incubation, the cell cultures were sent to a professional testing facility for sequencing confirmation to ensure the accuracy of the insert.

2024.8.28

The correctly sequenced plasmids were introduced into Nissle 1917 (EcN) competent cells and incubated overnight at 37°C. This step allows for plasmid replication and preparation for subsequent experiments.

2024.8.29

Well-growing cells were selected for expanded culture until they reached an OD600 of 0.6. Induction for green fluorescence expression was performed with a final concentration of 30 mM at 20°C, 30°C, and 37°C for 5 hours. Monitoring for fluorescence will determine the success of the expression under these conditions.

About Us：

Contact:

Address: 30 South Puzhu Road, Nanjing 211816, Jiangsu Province, China

![](https://static.igem.wiki/teams/5366/footer/f1.jpg)
![](https://static.igem.wiki/teams/5366/footer/f2.jpg)

![](https://static.igem.wiki/teams/5366/footer/f4.jpg)

---

© 2024 - Content on this site is licensed under  [Creative Commons Attribution 4.0 International license.](https://creativecommons.org/licenses/by/4.0/)

The repository used to create this website is available at [gitlab.igem.org/2024/njtech-china-a.](https://2024.igem.wiki/njtech-china-a/)

![](https://static.igem.wiki/teams/5366/home/home-picture/band.png)
[NJTech-China-A](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27home%27%29%20%7D%7D)

* [Project](#option1)

  [Description](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27description%27%29%20%7D%7D)
  [Contribution](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27contribution%27%29%20%7D%7D)
  [Protocol](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27protocol%27%29%20%7D%7D)
  [Engineering](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27engineering%27%29%20%7D%7D)
  [Experiments](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27experiments%27%29%20%7D%7D)
  [Notebook](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27notebook%27%29%20%7D%7D)
  [Results](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27results%27%29%20%7D%7D)
* [Part](#option2)

  [Part](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27part%27%29%20%7D%7D)
* [Model](#option3)

  [Model](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27model%27%29%20%7D%7D)
* [Team](#option4)

  [Members](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27members%27%29%20%7D%7D)
  [Attribution](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27attributions%27%29%20%7D%7D)
* [Human Practices](#option5)

  [Human Practice](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27human-practices%27%29%20%7D%7D)
  [Education](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27education%27%29%20%7D%7D)
  [Communication&Collaboration](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27communication%27%29%20%7D%7D)

# Engineering

* [The Research Process of New Enzyme Mining of Tagatose 4-Epimerase](#section1)
* [The Research Process of Soluble Expression](#section2)
* [I Fusion-promoting Tags](#section3)
* [II Molecular Chaperones](#section4)
* [III Fermentation Optimization](#section5)
* [The Research Process of Mutations](#section6)
* [Outlook](#section7)

The Research Process of New Enzyme Mining of Tagatose 4-Epimerase

Introduction

In this study, unknown functional proteins derived from *Chloroflexota bacterium*, *Pseudothermotoga hypogea DSM, Candidatus Aerophobetes bacterium*, and *Thermotogales bacterium* were identified to possess tagatose-4-epimerase activity through gene mining. These proteins were designated as MBC, AJC7, TET, and HDM. AJC7 exhibited the highest activity among the wild-type enzymes and was thus selected for further studies involving soluble expression and site-directed mutagenesis. The research process is detailed below.

Phylogenetic Tree Construction

Using the amino acid sequence UxaE from *Thermotoga petrophila* RKU-1 as a template, we performed a BLAST search against the non-redundant protein sequence database at the National Center for Biotechnology Information (NCBI). From the 100 sequences retrieved, 80 sequences with a similarity threshold below 70% were selected for further analysis. Multiple sequence alignment was conducted using the MEGA software, and a phylogenetic tree was constructed using the neighbor-joining method (Fig. 1).

![](https://static.igem.wiki/teams/5366/engineering/1.png)

Figure 1 The phylogenetic tree was constructed using the amino acid sequence UxaE derived from *Thermotoga petrophila* RKU-1 as a template for BLAST search

Protein Homology Modeling and Molecular Dynamics Simulation

1.Protein Structure Modeling

In this study, we used AlphaFold2 to perform structural modeling on the 80 selected sequences. AlphaFold2 is a state-of-the-art deep learning-based tool for predicting protein structures with high accuracy based on their amino acid sequences.

![](https://static.igem.wiki/teams/5366/engineering/2.png)

Figure 2 Modeling the structure of Alphafold2 (AJC7 as an example)

2.Molecular Docking

After completing protein homology modeling using AlphaFold2, we downloaded the structure file of D-tagatose from the PubChem website. In Discovery Studio 2019, we first opened the three-dimensional structure of the AJC7 protein. Then, in the tool browser, we selected "Receptor-Ligand Interactions" and clicked on "Define and Edit Binding Site" to define AJC7 as the receptor molecule. Next, we clicked "File > Open" to import the D-tagatose structure file downloaded from PubChem, and in the tool browser, we expanded "Small Molecules" under "Prepare or Filter Ligands > Prepare Ligands" to open the corresponding process parameter settings panel. After setting the "Input Ligands" parameters, we clicked "Run" to process the small molecule. Subsequently, we used the C-DOCKER module (a docking tool based on the CHARMM force field) to dock D-tagatose into the active site of AJC7. Candidate conformations were generated through random rigid-body rotation and simulated annealing. The structure of the protein-ligand complex underwent energy minimization using the CHARMM force field. Finally, we retrieved the lowest energy docking conformations using the C-DOCKER module and selected the substrate orientation with the lowest interaction energy with the ligand for further analysis. The conformations were ranked based on CHARMM energy, and the highest-scoring conformations were retained for further study.

3.Combined Free Energy Calculation

We calculated the binding free energy of the receptor-ligand complexes using the CHARMm-based energy function and an implicit solvent model. The binding energy between the receptor and ligand (ΔEBinding) is defined as EComplex = ELigand - EReceptor. To estimate these free energies, we minimized the ligand energy in the presence of the receptor using the steepest descent and conjugate gradient methods. The effective Born radii were computed using the Generalized Born Simple Switching (GBSW) implicit solvent model, replacing the costly molecular surface approximation with a smooth dielectric boundary combined with a van der Waals surface.

Using this approach, we calculated the binding free energy between the selected sequences and fructose, leading to the identification of four proteins with potential D-tagatose-4-epimerase activity

![](https://static.igem.wiki/teams/5366/engineering/03.png)

Figure 3 Binding self-energy between selected sequences and fructose

Considering the free energy data and phylogenetic tree analysis, four functional proteins with tagatose-4-epimerase activity were identified: MBC (MBC122911.1), AJC7 (AJC74158.1), TET (TET09249.1), and HDM (HDM70976.1).

Wild-type enzyme activity assay

Design

Four screened sequences with potential tagatose-4-epimerase activity were synthesized and ligated into the pET-28a(+) vector (Fig.4).

![](https://static.igem.wiki/teams/5366/engineering/4.png)

![](https://static.igem.wiki/teams/5366/engineering/5.png)

![](https://static.igem.wiki/teams/5366/engineering/6.png)

![](https://static.igem.wiki/teams/5366/engineering/7.png)

Figure 4 Plasmid maps of plasmids pET-28a(+)-MBP-AJC7, pET-28a(+)-NusA-AJC7, pET-28a(+)-TrxA-AJC7

Construction

Plasmids pET-28a(+)-AJC7, pET-28a(+)-HDM, pET-28a(+)-MBC, pET-28a(+)-TET, and pET-28a(+)-UxaE were transformed into *E. coli* BL21 (DE3) and cultured at 37°C for 14 hours. Individual colonies from the transformation were screened via colony PCR, and correct colonies were transferred to LB (Kan) medium for activation, amplification, and protein purification.

Test

The reaction was conducted with a final fructose concentration of 10 g/L and 1 mM Ni2+ as a catalyst. The volume of the pure enzyme required was determined based on protein concentration. The reaction was carried out at 70℃ for 5 hours, and products were analyzed using high-performance liquid chromatography (HPLC) (Fig.5).

![](https://static.igem.wiki/teams/5366/engineering/8.png)

Figure 5 Concentrations of tagatose produced by wild-type UxaE, AJC7, HDM, MBC, and TET under the same reaction conditions

HPLC results indicated that the activity of wild-type AJC7 significantly exceeded that of wild-type UxaE, producing approximately four times the tagatose concentration under identical catalytic conditions. AJC7 exhibited considerable potential as a tagatose-4-epimerase, warranting further investigation.

Study

During the experiments, AJC7 exhibited poor soluble expression, predominantly forming inclusion bodies (Fig. 6), complicating subsequent research on its enzymatic properties.

![](https://static.igem.wiki/teams/5366/engineering/9.png)

Figure 6 M: Protein marker; Lane 1: AJC7 precipitated Lane 2: AJC7 supernatant

To address this issue, we developed three strategies: fusion tags, molecular chaperones, and fermentation optimization.

The Research Process of Soluble Expression

I Fusion-promoting Tags

Introducation

Fusion-promoting tags can enhance the soluble expression of proteins by preventing aggregation of misfolded intermediates. We selected three tags to incorporate into AJC7:

1. Thioredoxin A (TrxA): Thermally stable and highly soluble.

2. Transcriptional Anti-Terminating Factor A (NusA):Prevents self-association of target proteins.

3. Maltose-binding protein (MBP): Known for its wide solubilization range and efficiency.

We inserted the gene of interest alongside the fusion-tagged DNA into a plasmid for transcription and translation, yielding a target protein attached to the fusion tag.

Design

AJC7 was combined with the three fusion-promoting tags via the linker GGGGSGGGGS, resulting in recombinant plasmids: pET-28a(+)-MBP-AJC7,pET-28a(+)-NusA-AJC7,and pET-28a(+)-TrxA-AJC7 (Fig.7).

![](https://static.igem.wiki/teams/5366/engineering/10.png)

![](https://static.igem.wiki/teams/5366/engineering/11.png)

![](https://static.igem.wiki/teams/5366/engineering/12.png)

Figure 7 Plasmid profiles of recombinant plasmids pET-28a(+)-MBP-AJC7, pET-28a(+)-NusA-AJC7 and pET-28a(+)-TrxA-AJC7

Construction

1.The fusion tag was amplified by PCR, and the pET-28a(+)-AJC7 vector was linearized using reverse PCR, followed by gel recovery of the correct fragment.

2.The fusion-promoting tag fragment underwent homologous recombination with the vector, and heat shock transformation was performed. Colonies were cultured, and colony PCR confirmed the presence of recombinant plasmids (Fig. 8).

3.Verified colonies were sequenced, and those with correct sequences underwent expression induction. Following induction, the cell lysate was analyzed by SDS-PAGE. The strains modified with the fusion-promoting tag were designated TrxA-AJC7, NusA-AJC7, and MBP-AJC7,respectively.

Test

![](https://static.igem.wiki/teams/5366/engineering/13.png)

Figure 8 Nucleic acid gel diagram of colony PCR

![](https://static.igem.wiki/teams/5366/engineering/014.png)

![](https://static.igem.wiki/teams/5366/engineering/15.jpg)

Figure 9 SDS-PAGE of recombinant AJC7 with a fusion-promoting tag (M: protein marker; Lane 1: precipitation of AJC7; Lane 2: supernatant of AJC7; Lane 3: precipitation of TrxA-AJC7; Lane 4: supernatant of TrxA-AJC7; Lane 5: precipitation of NusA-AJC7; Lane 6: Supernatant of NusA-AJC7; Lane 7: precipitation of MBP-AJC7; Lane 8: Supernatant of MBP-AJC7)

The molecular weights of AJC7, TrxA-AJC7, NusA-AJC7, and MBP-AJC7 are 60.4 kDa, 70.4 kDa, 113.4 kDa, and 99.0 kDa, respectively. As illustrated in Fig. 8, there was minimal difference between the supernatant and precipitation of the original AJC7 (lanes 1 and 2) in the cell lysate, suggesting that the soluble expression of the target protein was limited. The concentration of the 70.4 kDa protein band (lane 4) in the supernatant was significantly increased, while the proportion of the precipitated protein band (lane 3) was reduced. Similarly, the concentration of the 113.4 kDa protein band (lane 6) in the supernatant also showed a significant increase, accompanied by a decrease in the precipitated protein band. The 99.0 kDa protein band (lane 8) exhibited a notable increase in the supernatant, with a corresponding reduction in the density of the precipitated protein band. Induced at 20℃ for 16 hours, further densitometric analysis of the protein gel was conducted to quantify the increase in protein expression, and the results are summarized as follows:

![](https://static.igem.wiki/teams/5366/engineering/16.jpg)

Figure 10 Histogram of densitometry data of SDS-PAGE gel supernatant and precipitated protein bands of bacterial cell breakage fluid before and after fusion-promoting tag attachment

![](https://static.igem.wiki/teams/5366/engineering/17.jpg)

Figure 11 Percentage comparison of densitometry data for bacterial cell breakage, supernatant, and precipitated protein bands before and after fusion-promoting tag attachment

Figures 10 and 11 demonstrate that under specific induction conditions, the solubility of the protein was significantly enhanced by NusA, TrxA, and MBP, increasing by 6.86, 6.79, and 6.92 times, respectively, compared to AJC7. Additionally, the density of the protein bands in the precipitate was greatly reduced. This provides further validation for the inference that incorporating the fusion tag effectively enhances the solubility of the target protein.

Study

The solubility of proteins in *E. coli* improved significantly with fusion tags at the N-terminus of AJC7. However, additional proteases are required to cleave the fusion protein for pure enzyme recovery. To enhance expression and solubility while maintaining practicality, we explored molecular chaperones.

II Molecular Chaperones

Introduction

Molecular chaperones facilitate proper protein folding and reduce inclusion body formation without compromising protein activity. A Chaperone Plasmid Set (including pG-KJE8, pGro7, pKJE7, pG-Tf2, and pTf16) was co-introduced into *E. coli* BL21 (DE3) with the AJC7 plasmid, aiming to improve soluble protein recovery through synergistic folding assistance.

Design

Five chaperone plasmids pG-KJE8, pGro7, pKJE7, pG-Tf2 and pTf16 were co-expressed with pET-28a(+)-AJC7 plasmid, respectively, to promote the correct folding of exogenous proteins in prokaryotic cells and increase the solubility of exogenous proteins, in order to obtain a large number of soluble proteins and explore the biological functions of the proteins.
The modified strain pairs were named pG-KJE8/AJC7, pGro7/AJC7, pKJE7/AJC7, pG-Tf2/AJC7, pTf16/AJC7.

Construction

In this study, five chaperone plasmids were co-transformed into *E. coli* BL21 (DE3) along with the pET-28a(+)-AJC7 plasmid using thermomechanical transformation methods. The transformed cells were initially screened on plates containing kanamycin (Kan) and chloramphenicol (Cm) to ensure successful plasmid incorporation. Single colonies that tested positive via colony PCR were induced for protein expression. Post-induction, the cells were disrupted, and both the supernatant and precipitate were analyzed using SDS-PAGE.

Test

In this study, five chaperone plasmids were co-transformed into *E. coli* BL21 (DE3) along with the pET-28a(+)-AJC7 plasmid using thermomechanical transformation methods. The transformed cells were initially screened on plates containing kanamycin (Kan) and chloramphenicol (Cm) to ensure successful plasmid incorporation. Single colonies that tested positive via colony PCR were induced for protein expression. Post-induction, the cells were disrupted, and both the supernatant and precipitate were analyzed using SDS-PAGE.

![](https://static.igem.wiki/teams/5366/engineering/18.png)

Figure 12 SDS-PAGE co-expressed by chaperone and pET-28a(+)-AJC7 (M: protein marker; Lane 1: precipitation of AJC7; Lane 2: supernatant of AJC7; Lane 3: precipitation of pG-KJE8/AJC7; Lane 4: supernatant of pG-KJE8/AJC7; Lane 5: precipitation of pGro7/AJC7; Lane 6: supernatant of pGro7/AJC7; Lane 7: precipitation of pKJE7/AJC7; Lane 8: supernatant of pKJE7/AJC7; Lane 9: precipitation of pG-Tf2/AJC7; Lane 10: supernatant of pG-Tf2/AJC7; Lane 11: precipitation of pTf16/AJC7; Lane 12: supernatant of pTf16/AJC7)

Subsequent analysis of the protein bands on the gel was conducted using densitometry (see Figures 13-14).

![](https://static.igem.wiki/teams/5366/engineering/19.jpg)

Figure 13 Histogram of densitometry analysis data of SDS-PAGE gel supernatant and precipitated protein bands of bacterial cell breakage fluid co-expressed with chaperone proteins

![](https://static.igem.wiki/teams/5366/engineering/20.jpg)

Figure 14 Comparison of the percentage of densitometry data of bacterial cell breakage, supernatant and precipitated protein bands co-expressing the target gene and chaperone protein

The results indicated that the solubility of the target protein pG-Tf2/AJC7 increased by 2.2 times compared to AJC7, as evidenced by the higher density of protein bands in the supernatant and a reduction in the precipitate. This suggests that the inclusion of chaperone proteins significantly enhanced the solubility of the target protein.

Study

Different combinations of chaperones appear to influence specific classes of proteins, indicating that their effectiveness in enhancing soluble expression can vary based on the target protein and the expression conditions. This study aims to explore whether modifications in culture conditions can further improve protein solubility. The focus will be on optimizing inducers, induction temperatures, and media composition.

III Fermentation Optimization

Introduction

Induction temperature and IPTG concentration are critical factors that can influence the expression level and folding quality of target proteins, as well as the overall growth of *E. coli*. The use of self-inducing media can mitigate cytotoxic effects on the expressed protein, as well as reduce contamination risks by eliminating the need to add inducers midway through the process. This study aims to optimize fermentation parameters including temperature, IPTG concentration, and medium composition.

Design

1.Optimal Temperature

*E. coli* BL21 (DE3) was cultured at 37℃ with shaking at 200 rpm. Once the optical density (OD) reached 0.6 at 600 nm, IPTG was added to a final concentration of 0.5 mM. The cultures were then incubated at varying temperatures (16℃, 20℃, 30℃, and 37℃) for 16 hours, shaking at 200 rpm, to induce protein expression.

2.Optimal IPTG Concentration

The same initial conditions were maintained at 37 ℃ with shaking. Upon reaching OD 0.6, cultures received IPTG at varying final concentrations (0.1 mM, 0.2 mM, 0.5 mM, and 1 mM). The cultures were then incubated at 20 ℃ for 16 hours with shaking at 200 rpm to induce protein expression.

3. Research on Self-Inducing Medium

To assess the effects of different carbon sources on protein expression, 16 variations of self-inducing medium were prepared, incorporating different combinations of carbon sources along with 10 g/L of NaCl. The recombinant *E. coli* BL21 (DE3) was inoculated and cultured at 37℃ for 3 hours, then shifted to 20℃ for 20 hours, shaking at 200 rpm to induce enzyme expression. As a control, cultures were induced with a final IPTG concentration of 0.1 mM once OD 0.6 was achieved.

Table 1 Recipe table of self-inducing medium

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Horizontal factors | Tryptone（g/L） | Yeast powder（g/L） | Glycerol（g/L） | Glucose（g/L） | Lactose（g/L） |
| 1 | 5 | 5 | 3 | 0.3 | 1 |
| 2 | 10 | 10 | 5 | 0.5 | 2 |
| 4 | 20 | 20 | 9 | 0.9 | 4 |

Table 2 Table of factor levels of self-inducing medium

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Experimental group factors | Tryptone | Yeast powder | Glycerol | Glucose | Lactose |
| 1 | 1 | 1 | 1 | 1 | 1 |
| 2 | 1 | 2 | 2 | 2 | 2 |
| 3 | 1 | 20 | 3 | 3 | 3 |
| 4 | 1 | 4 | 4 | 4 | 4 |
| 5 | 2 | 1 | 2 | 3 | 4 |
| 6 | 2 | 2 | 1 | 4 | 3 |
| 7 | 2 | 3 | 4 | 1 | 2 |
| 8 | 2 | 4 | 3 | 2 | 1 |
| 9 | 3 | 1 | 3 | 4 | 2 |
| 10 | 3 | 2 | 4 | 3 | 1 |
| 11 | 3 | 3 | 1 | 2 | 4 |
| 11 | 3 | 3 | 1 | 2 | 4 |
| 12 | 3 | 4 | 2 | 1 | 3 |
| 13 | 4 | 1 | 4 | 2 | 3 |
| 14 | 4 | 2 | 3 | 1 | 4 |
| 15 | 4 | 3 | 2 | 4 | 1 |
| 16 | 4 | 4 | 1 | 3 | 2 |

Test

1.Optimal Temperature for Induction

![](https://static.igem.wiki/teams/5366/engineering/21.jpg)

Figure 15 SDS-PAGE of recombinant AJC7 induced at different temperatures（M：marker； Lane 1: precipitation of enzyme AJC7 after induction at 16℃; Lane 2: precipitation of enzyme AJC7 after induction at 20℃; Lane 3: precipitation of enzyme AJC7 after induction at 30℃; Lane 4: precipitation of enzyme AJC7 after induction at 37℃; Lane 5: supernatant of enzyme AJC7 after induction at 16 ℃; Lane 6: supernatant of enzyme AJC7 after induction at 20℃; Lane 7: supernatant of enzyme AJC7 after induction at 30℃; Lane 8: supernatant of enzyme AJC7 after induction at 37℃)

The molecular weight of AJC7 is 60.375 kDa, as shown in Figure 14. After induction at 16 ℃, the concentration of protein bands in the supernatant (lane 5) was higher than that in the precipitation (lane 1). Similarly, after induction at 20 ℃, the concentration of protein bands in the supernatant (lane 6) exceeded that of the precipitation (lane 2). However, after induction at 30 ℃, the concentration of protein bands in the supernatant (lane 7) was lower than in the precipitation (lane 3). After induction at 37 ℃, the concentration of protein bands in the supernatant (lane 8) was also lower than that in the precipitation (lane 4).

Induced at various temperatures for 16 hours, further densitometric analysis of the protein gel was conducted to quantify the increase in protein expression, and the results are summarized as follows:

![](https://static.igem.wiki/teams/5366/engineering/22.png)

Figure 16 Histogram of densitometry analysis data of SDS-PAGE gel supernatant and precipitated protein bands of bacterial cell breakage fluid after induction at different temperatures

![](https://static.igem.wiki/teams/5366/engineering/23.png)

Figure 17 Comparison of optical densitometry data percentages of bacterial cell breakage, supernatant and precipitated protein bands after different temperature induction

Figures 16 and 17 indicate that the soluble expression of AJC7 protein increases at both 16 ℃ and 20 ℃, with a 1.24-fold higher expression at 16 ℃ compared to that at 20 ℃. Additionally, the density of the protein bands in the precipitate is significantly reduced. This provides further validation for the inference that selecting an appropriate induction temperature can effectively enhance the solubility of the target protein, with 16 ℃ identified as the optimal temperature for induction.

2.Optimal IPTG Concentration

![](https://static.igem.wiki/teams/5366/engineering/023.png)

Figure 18 SDS-PAGE of recombinant AJC7 induced by different concentrations of IPTG (M: protein marker; Lane 1: precipitation of enzyme AJC7 after 0.1 mM induction, lane 2: precipitation of enzyme AJC7 after 0.2 mM induction, lane 3: precipitation of enzyme AJC7 after 0.5 mM induction, lane 4: precipitation of enzyme AJC7 after 1 mM induction, lane 5: supernatant of enzyme AJC7 after 0.1 mM induction, lane 6: supernatant of enzyme AJC7 after 0.2 mM induction, lane 7: supernatant of enzyme AJC7 after 0.5 mM induction, lane 8: supernatant of enzyme AJC7 after 1 mM induction)

AJC7 has a molecular weight of 60.375 kDa. As shown in Figure 18, the concentration of protein bands in the supernatant (lane 5) of the cell disruption solution after 0.1 mM IPTG induction is higher than that in the precipitation (lane 1). Similarly, after 0.2 mM IPTG induction, the concentration of protein bands in the supernatant (lane 6) exceeds that of the precipitation (lane 2). This trend continues with 0.3 mM IPTG induction, where the concentration in the supernatant (lane 7) is also higher than in the precipitation (lane 3). After 1 mM IPTG induction, the concentration of protein bands in the supernatant (lane 8) remains greater than that in the precipitation (lane 4).

Induced at 20 ℃with varying IPTG concentrations for 16 hours, further densitometric analysis of the protein gel was conducted to quantify the increase in protein expression, and the results are summarized as follows:

![](https://static.igem.wiki/teams/5366/engineering/024.jpg)

Figure 19 Histogram of densitometry data of SDS-PAGE gel supernatant and precipitated protein bands of bacterial cell breakage fluid induced by different concentrations of IPTG

![](https://static.igem.wiki/teams/5366/engineering/25.png)

Figure 20 Percentage comparison of densitometry data of bacterial cell breakage, supernatant and precipitated protein bands after IPTG induction at different concentrations

![](https://static.igem.wiki/teams/5366/engineering/26.png)

Figures 19 and 20 illustrate that the solubility of the protein increased by 1.39-fold, 1.43-fold, and 1.48-fold following induction with IPTG at concentrations of 0.2 mM, 0.5 mM, and 1 mM, respectively. Additionally, the density of the protein bands in the precipitate was significantly reduced. Therefore, at an IPTG concentration of 0.5 mM, protein expression was found to be the most abundant, resulting in optimal solubility.

3.Self-inducing Medium

A

![](https://static.igem.wiki/teams/5366/engineering/27.png)

B

![](https://static.igem.wiki/teams/5366/engineering/28.png)

C

![](https://static.igem.wiki/teams/5366/engineering/29.png)

Figures 21 SDS-PAGE of recombinant AJC7 after induction in autoinduction medium with different combinations of carbon sources(M: protein marker; Lane C: supernatant of enzyme AJC7 after induction in normal LB control group; Lanes 1-16: supernatant of enzyme AJC7 after induction in experimental groups 1-16; Lane C': precipitation of enzyme AJC7 after induction in the normal LB control group, and lanes 1'-16': precipitation of enzyme AJC7 after induction in experimental groups 1-16)

The molecular weight of AJC7 is 60.375 kDa, as shown in Figure 21. The supernatants of the cell disruption solutions after induction in experimental groups 1, 2, 14, 15, and 16 exhibited higher concentrations of protein bands compared to those induced with standard LB medium, with the highest concentration observed in the supernatant of experimental group 16. Additionally, the concentration of protein bands in the cell disruption solutions for experimental groups 2, 14, 15, and 16 was lower than that observed after induction with ordinary LB.

Induced at 20°C for 20 hours, further densitometric analysis of the protein gel was conducted to quantify the increase in protein expression, and the results are summarized as follows:

![](https://static.igem.wiki/teams/5366/engineering/30.png)

Figures 22 Histogram of densitometry data of bacterial cell breakage fluid SDS-PAGE gel supernatant and precipitated protein bands after induction in autoinduction medium with different combinations of carbon sources(0: common LB control group; 1-16: Test group 1-16)

![](https://static.igem.wiki/teams/5366/engineering/31.png)

Figures 23 Comparison of optical density analysis data of bacterial cell breakage, supernatant, and precipitated protein bands after induction in autoinduction medium with different combinations of carbon sources(0: common LB control group; 1-16: Test group 1-16)

Figures 22 and 23 demonstrate that the solubility of the target protein increased by 1.82 times, 2.33 times, 2.15 times, 2.36 times, and 2.15 times, respectively, in experimental groups 1, 2, 14, 15, and 16 compared to ordinary LB medium. Additionally, the density of the protein bands in the precipitate was significantly reduced.

Study

In the field of synthetic biology, selecting the optimal system requires careful consideration of several factors. The optimization of fermentation conditions has established a foundation for the efficient soluble expression of AJC7 in *E. coli*. Through extensive literature research, we found that mutations are a common method to enhance the activity of the enzyme AJC7. Consequently, in the subsequent experimental process, we decided to employ a mutagenesis strategy to increase the activity of AJC7.

The Research Process of Mutations

Introducation

In this study, the UxaE amino acid sequence from *Thermus neapolitana* served as a template for gene mining. We identified four enzyme sequences with potential tagatose-4-epimerase activity, named MBC, HDM, TET, and AJC7, based on phylogenetic analysis and binding free energy considerations. Given their soluble and wild-type enzyme activities, AJC7 was selected for further investigations involving single-point and iterative mutations. The catalytic mechanism of tagatose-4-epimerase, which converts fructose to tagatose, has been well-characterized (Fig. 24). Focusing on the structure and function of the AJC7 protein, we constructed a series of mutants: S125D, T181A, H342L, I129T, L140P, and various combinations thereof.

![](https://static.igem.wiki/teams/5366/engineering/32.jpg)

Figures 24 Catalytic mechanism of tagatose-4-epimerase catalyzing fructose production of tagatose[1]

![](https://static.igem.wiki/teams/5366/engineering/33.png)

Figures 25. （A）Docking model of D-fructose in the H342I mutant,（B）Docking model of D-fructose in the I129T mutant,（C）Docking model of D-fructose in the L140T mutant,（D）Docking model of D-fructose in the S125D mutant,（E）Docking model of D-fructose in the T181A mutant.

Table 3 Reasons for mutation site selection

|  |  |
| --- | --- |
| Mutation point | Reasons for choosing |
| S125D | The conversion of fructose to D-tagatose begins with the formation of a glyceraldehyde intermediate. When serine is mutated to aspartic acid, the carboxyl group of aspartic acid can interact with the terminal aldehyde group of the glyceraldehyde intermediate, facilitating the protonation of the aldehyde and thereby aiding in the catalysis and production of D-tagatose. Furthermore, the negatively charged aspartic acid may enhance interactions with the positively charged substrate and alter the charge distribution in the binding pocket, thereby increasing the catalytic activity of the enzyme. |
| T181A | By mutating from a large amino acid to a smaller one and from a polar to a non-polar amino acid, the mutation increases the hydrophobicity within the active pocket, thereby contributing to the stability of the protein. |
| H342L | Replacing the positively charged histidine with the uncharged and more hydrophobic leucine alters the charge properties of the amino acid residue, reconfiguring the charge distribution within the substrate pocket. This enhances interactions with the substrate. Additionally, the increased hydrophobicity within the active pocket improves the enzyme's stability. |
| I129T | After replacing the nonpolar amino acid isoleucine with the polar amino acid threonine, the hydroxyl group in the threonine side chain can form a new hydrogen bond interaction with serine at position 125. This enhanced interaction may increase the enzyme's stability and alter its conformation, thereby creating a more favorable environment in the active pocket for substrate binding through a transmission effect, ultimately improving catalytic efficiency. |
| L140P | Proline has a cyclic structure, and its unique secondary amine limits the rotational freedom of the residue. This restriction leads to changes in the enzyme's three-dimensional structure, which, through a transmission effect, alters the interactions between the active pocket and the substrate, thereby enhancing catalytic activity. |

Design 1

We designed primers for point mutations S125D, T181A, H342L, I129T, and L140P, using pET-28a(+)-AJC7 as a template to amplify single-point mutants (Fig. 26). This design was pivotal for assessing the impact of each mutation on enzyme activity.

![](https://static.igem.wiki/teams/5366/engineering/34.jpg)

Figures 26 Point mutation localization and primer design

Construction 1

![](https://static.igem.wiki/teams/5366/engineering/35.png)

Figures 27 Nucleic acid gel diagram of colony PCR

The PCR mutation protocol is outlined in the experimental section. Following PCR amplification, the products underwent demethylation using DpnI. Verification of the PCR products involved nucleic acid gel electrophoresis. The successful constructs were then transformed into *E. coli* BL21 (DE3) and incubated at 37°C for 14 hours. Individual colonies were picked for colony PCR, as depicted in Fig. 27. After confirmation through electrophoresis, the positive colonies were transferred to LB (Kan) liquid medium. Subsequent sequencing confirmed the successful construction of all five single-point mutants.

Test 1

We activated and cultured both the mutant strains and the wild-type strain, followed by a series of purification steps to extract the target protein using methods outlined in the experimental section. The volume of purified enzyme solution for the 500µL reaction system was determined based on protein concentration. A final fructose concentration of 100 g/L was established, with 10µL of Ni2+ as a catalyst. Reactions were conducted at 70°C for 5 hours, and the products were analyzed via high-performance liquid chromatography (HPLC) (Fig. 28).

The results demonstrated that the concentrations of fructose from the mutants—S125D, T181A, H342L, I129T, and L140P—were higher than those from the wild-type AJC7. Notably, the H342L, I129T, and L140P mutations showed less pronounced improvements. The S125D mutant exhibited a catalytic efficiency that was twice that of the wild type under identical reaction conditions and substrate concentrations.

![](https://static.igem.wiki/teams/5366/engineering/36.png)

Figures 28 The concentrations of tagatose produced in the system after WT, S125D, T181A, H342L, I129T, and L140P reacted with 100 g/L substrate fructose for 5 h

The results showed that the concentrations of AJC7 after five single-point mutations, namely S125D, T181A, H342L, I129T and L140P, were higher than those of wild-type AJC7. The improvement effect of H342L, I129T, and L140P is not obvious. The catalytic efficiency of the S125D mutant was twice as high as that of the wild type under the same reaction conditions and substrate concentration.

Study 1

Because the catalytic efficiency of S125D was significantly improved compared with WT type, we decided to use S125D as a template to construct two-point mutants of S125D/T181A, S125D/H342L, S125D/I129T, and S125D/L140P to further improve the enzyme activity.

Design 2

The three mutation points, T181A, H342L, and L140P, are located at a considerable distance from S125D. Therefore, pET-28a(+)-AJC7-S125D can be utilized as a template for the construction of the double mutants S125D/T181A, S125D/H342L, and S125D/L140P using the primers previously employed for single-point mutations in Design 1. However, due to the close proximity of I129T to S125D, the primers for the I129T mutation were redesigned using pET-28a(+)-AJC7-S125D as a template to create the S125D/I129T mutant.

Construction 2

The same methodology as outlined in Construction 1 was applied to generate the double mutants S125D/T181A, S125D/H342L, S125D/I129T, and S125D/L140P.

![](https://static.igem.wiki/teams/5366/engineering/37.jpg)

Figures 29 Nucleic acid gel diagram of colony PCR

Test 2

Same method as [Test 1].

![](https://static.igem.wiki/teams/5366/engineering/38.jpg)

Figures 30 The concentrations of tagatose in wild-type, S125D, S125D/T181A, S125D/H342L, S125D/I129T, and S125D/L140P after reaction with 100g/L fructose substrate for 5 h, respectively

The results indicated that the concentrations of AJC7 following the mutations S125D/T181A, S125D/H342L, S125D/I129T, and S125D/L140P were significantly higher than those of both wild-type AJC7 and S125D. Notably, the S125D/T181A double mutant exhibited the most substantial increase in AJC7 enzyme activity, with its catalytic efficiency nearly doubling that of wild-type AJC7.

Study 2

Compared to wild-type AJC7, the enzyme activity of the S125D/T181A double mutant was significantly enhanced. Consequently, we decided to use S125D/T181A as a template to construct additional superimposed mutants, specifically S125D/T181A/I129T, S125D/T181A/L140P, and S125D/T181A/H342L, in order to further improve enzyme activity.

Design 3

The single-point mutagenesis primers used in Design 1 were directly applied in this phase.

Construction 3

Construction 1 was utilized to successfully generate the three-point mutants S125D/T181A/I129T, S125D/T181A/L140P, and S125D/T181A/H342L.

Test 3

The procedures were consistent with those outlined in Test 1.

![](https://static.igem.wiki/teams/5366/engineering/39.jpg)

Figures 31 Nucleic acid gel diagram of colony PCR

Test 3

The procedures were consistent with those outlined in Test 1.

![](https://static.igem.wiki/teams/5366/engineering/40.png)

Figures 32 The concentrations of tagatose in wild-type, S125D, S125D/T181A, S125D/T181A/I129T, and S125D/T181A/H342L reacted with 100 g/L fructose substrate for 5 h, respectively

The results demonstrated that the concentrations of AJC7 following the S125D/T181A/I129T and S125D/T181A/H342L three-point mutations were higher than those observed for wild-type AJC7, S125D, and S125D/T181A. However, the S125D/T181A/L140P three-point mutant did not yield a significant improvement in enzyme activity. Among the mutants, the S125D/T181A/I129T exhibited the highest increase in AJC7 enzyme activity, with a catalytic efficiency nearly three times greater than that of wild-type AJC7.

Study 3

The S125D/T181A/I129T three-point mutant demonstrated further improvements in enzyme activity. Consequently, we utilized S125D/T181A/I129T as a template for saturation mutagenesis, constructing the S125D/T181A/I129T/L140P four-point mutant and the S125D/T181A/I129T/L140P/H342L five-point mutant to enhance enzyme activity further.

Design 4

We directly employed the single-point mutagenesis primers outlined in [Design 1].

Construction 4

The same method as in [Construction 1] was used to construct S125D/T181A/I129T/L140P four-point mutants and S125D/T181A/I129T/L140P/H342L five-point mutants.

![](https://static.igem.wiki/teams/5366/engineering/41.jpg)

Figures 33 Nucleic acid gel diagram of colony PCR (4-point mutation on the left and 5-point mutation on the right)

Test 4

Same method as [Test 1].

![](https://static.igem.wiki/teams/5366/engineering/42.png)

Figures 34 The concentrations of tagatose in WT, S125D, S125D/T181A, S125D/T181A/I129T, S125D/T181A/I129T/L140PS125D/T181A/I129T/L140P/H342L after reacting with 100g/L fructose substrate for 5 h

The results indicated that the S125D/T181A/I129T/L140P four-point mutant and the S125D/T181A/I129T/L140P/H342L five-point mutant exhibited improved enzyme activity compared to wild-type AJC7. However, the enzyme activity of the S125D/T181A/I129T was found to be unsatisfactory relative to the S125D/T181A mutant.

Study 4

The enhancements in enzyme activity for the S125D/T181A/I129T/L140P four-point mutant and S125D/T181A/I129T/L140P/H342L five-point mutant did not meet our expectations. In future experiments, we will explore additional mutation sites to facilitate further superimposed mutations of the S125D/T181A/I129T three-point mutants to improve enzyme activity, or we may pursue directed evolution of AJC7 to enhance its enzymatic performance. To gain deeper insights into the S125D/T181A/I129T three-point mutants, we conducted further assays on their enzymatic properties and kinetic parameters.

The Research Process of Determination of Enzymatic Properties and Enzyme Kinetic Parameters

Introduction

In this study, we focused on the iterative mutation of the AJC7 enzyme, specifically identifying the three-point mutant S125D/T181A/I129T, which exhibited a nearly threefold increase in the conversion rate of fructose to tagatose. To further understand the enzymatic properties and industrial applicability of this mutant, we investigated the optimal pH and temperature for its activity. Additionally, we examined the relationship between the enzyme's conversion rate over time and determined its kinetic parameters, particularly the Michaelis constant (Km), to assess the enzyme's activity and affinity for the substrate.

Design

1.Enzymatic Properties Studies

a.In order to study the optimal pH of the reaction, the enzyme solution was reacted in 50mM buffer at pH 3~11 (in which the concentration of fructose in the substrate was 100g/L and the final concentration of the pure enzyme solution was 0.1mg/ml), 70 °C for 3h, containing 1 mM Ni2+. The buffer system is as follows: pH3-5: citric acid-sodium citrate; pH6-7：Na2HPO4-NaH2PO4;pH8-9：Tris-HCl; pH10-11：NaOH-NaH2PO4.

b. The optimal temperature for the enzyme reaction was assessed by incubating the reaction mixtures containing 100 g/L fructose, 0.1 mg/mL enzyme, and 1 mM Ni2+ in Tris-HCl buffer (pH 9.0) for 2 hours at varying temperatures: 50°C, 60°C, 70°C, 80°C, and 90°C. After incubation, the reaction products were analyzed via HPLC, and an optimal temperature curve was generated to determine the temperature at which the enzyme activity was maximized.

c. To evaluate the maximum conversion rate of the S125D/T181A/I129T mutant, we set up a reaction system with a final concentration of 23 mg/mL of the pure enzyme in a 500µL volume, along with a fructose concentration of 100 g/L and 1 mM Ni2+. The buffer was adjusted to 50 mM Tris-HCl at pH 9.0. The reaction was conducted at 70℃ in a metal shaker, and samples were taken every 20 minutes over a total period of 180 minutes. After the reaction, the concentrations of tagatose produced and remaining fructose were quantified using HPLC, enabling us to calculate the conversion rate.

2. Enzyme Kinetic Parameter Determination

To determine the Michaelis constant (Km) of the optimal mutant S125D/T181A/I129T of AJC7, a concentration gradient of fructose was established, with concentrations of 5 g/L, 10 g/L, 20 g/L, 40 g/L, 50 g/L, 60 g/L, 80 g/L, and 100 g/L. The final enzyme concentration was set at 0.1 mg/mL, and 1 mM Ni2+ was added as a catalytic metal ion. The remaining volume was adjusted to 50 mM Tris-HCl buffer at pH 8.0. The reaction was conducted at 70°C in a metal shaker for 20 minutes. After the reaction, the yield of tagatose and the residual fructose concentration were analyzed using high-performance liquid chromatography (HPLC), and the conversion rate was subsequently calculated.

Test

Enzymatic Properties Studies

a.Calculate the relative activity under other pH conditions separately with a maximum activity of 100% at pH 9.0.

![](https://static.igem.wiki/teams/5366/engineering/43.png)

Figures 35 The relative activities of AJC7-S125D/T181A/I129T at different pH.
The activity at pH 9.0 was set as 100%

Figure 35 illustrates that AJC7 exhibits minimal activity in catalyzing the conversion of fructose to tagatose at pH levels between 4.0 and 5.0. However, enzyme activity increases with rising pH values within the range of 6.0 to 9.0, reaching a maximum at pH 9.0. Beyond this point, specifically from pH 9.0 to 11.0, enzyme activity begins to decline. These findings suggest that AJC7 is more effective in facilitating the conversion of fructose to tagatose in a weakly alkaline environment.

b. The maximum activity observed at 70°C was designated as 100%, and relative activity at other temperature conditions was calculated accordingly (Fig. 36).

![](https://static.igem.wiki/teams/5366/engineering/44.png)

Figures 36 AJC7-S125D/T181A/I129T triple mutant enzyme activity as a function of reaction temperature

As illustrated in Fig. 36, at the optimal pH of 9.0, the enzyme activity of the AJC7-S125D/T181A/I129T three-point mutant increased gradually with rising temperatures within the range of 50–70°C, peaking at 70°C. Beyond this temperature, enzyme activity began to decline. Therefore, the optimal reaction temperature for the AJC7-S125D/T181A/I129T three-point mutant in catalyzing the conversion of fructose to tagatose is determined to be 70°C

c. To determine the conversion rate of tagatose-4-epimerase AJC7, the substrate conversion of the optimal mutant was assessed under optimal reaction conditions (70°C, pH 9.0, and 1 mmol/L Ni2+).

![](https://static.igem.wiki/teams/5366/engineering/45.png)

Figures 37 Conversion rate of the best AJC7 mutant under optimal conditions

As shown in Figure 36, when the final enzyme concentration was 23 mg/mL, the optimal mutant of AJC7 catalyzed the conversion of 100 g/L fructose to produce 37 g/L tagatose within 120 minutes, resulting in a conversion rate of 37% and a yield of 18.5 g/(L·h). Notably, at 80 minutes, 33 g/L of tagatose was generated, corresponding to a conversion rate of 33% and a peak yield of 24.1 g/(L·h).

Enzyme Kinetic Parameter Determination

![](https://static.igem.wiki/teams/5366/engineering/46.png)

Figures 38 Nonlinear regression equations for substrate concentration and reaction rate

The values of Km (mM) and Vmax (h/mM) were determined by Origin soft nonlinear regression with Vm of 27.3086 and Km of 99.805. It can be seen that the best mutant of AJC7, S125D/T181A/I129T, has a good affinity with the substrate.

Study

The conversion rate of the most effective mutant of AJC7, S125D/T181A/I129T, has improved; however, it still does not meet the requirements for industrial production. To address this limitation, we aim to obtain mutant strains with even higher conversion rates through directed evolution. Before proceeding, it is essential to develop an efficient and rapid high-throughput screening method to replace traditional high-performance liquid chromatography. Consequently, we focused on designing a biosensor that exhibits specific green fluorescence in response to tagatose.

Outlook

Currently, the developed biosensor demonstrates a specific response to tagatose. However, due to time constraints during the competition and certain challenges encountered in the experimental process, the sensor's sensitivity to changes in tagatose concentration is limited, hindering the screening of high tagatose-4-epimerase activity based on fluorescence values. In future work, we aim to construct new plasmids and mutate promoters to enhance sensitivity. We also plan to explore and improve novel methodologies, ultimately striving to provide a practical tool for high-throughput screening. This work will also offer valuable insights and components for the iGEM team to employ high-throughput screening methods for directed evolution in future projects

About Us：

Contact:

Address: 30 South Puzhu Road, Nanjing 211816, Jiangsu Province, China

![](https://static.igem.wiki/teams/5366/footer/f1.jpg)
![](https://static.igem.wiki/teams/5366/footer/f2.jpg)

![](https://static.igem.wiki/teams/5366/footer/f4.jpg)

---

© 2024 - Content on this site is licensed under  [Creative Commons Attribution 4.0 International license.](https://creativecommons.org/licenses/by/4.0/)

The repository used to create this website is available at [gitlab.igem.org/2024/njtech-china-a.](https://2024.igem.wiki/njtech-china-a/)

![](https://static.igem.wiki/teams/5366/home/home-picture/band.png)
[NJTech-China-A](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27home%27%29%20%7D%7D)

* [Project](#option1)

  [Description](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27description%27%29%20%7D%7D)
  [Contribution](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27contribution%27%29%20%7D%7D)
  [Protocol](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27protocol%27%29%20%7D%7D)
  [Engineering](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27engineering%27%29%20%7D%7D)
  [Experiments](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27experiments%27%29%20%7D%7D)
  [Notebook](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27notebook%27%29%20%7D%7D)
  [Results](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27results%27%29%20%7D%7D)
* [Part](#option2)

  [Part](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27part%27%29%20%7D%7D)
* [Model](#option3)

  [Model](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27model%27%29%20%7D%7D)
* [Team](#option4)

  [Members](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27members%27%29%20%7D%7D)
  [Attribution](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27attributions%27%29%20%7D%7D)
* [Human Practices](#option5)

  [Human Practice](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27human-practices%27%29%20%7D%7D)
  [Education](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27education%27%29%20%7D%7D)
  [Communication&Collaboration](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27communication%27%29%20%7D%7D)

# Experiments

* [Preparation of LB Medium](#section1)
* [Construction of Soluble Expression Module Plasmids](#section2)
* [Linearization of pET-28a(+)-AJC7 Vectors](#section3)
* [Gibson Assembly and Heat Shock Transformation](#section4)
* [Dual Plasmid Transformation](#section5)
* [Product Purification](#section6)
* [Construction Of Mutation Module Plasmids](#section7)
* [Colony PCR and Sequencing](#section8)
* [Cell Cultivation and Induction](#section9)
* [Cell Fragmentation and Protein Harvesting](#section10)
* [Protein Purification](#section11)
* [SDS-PAGE Electrophoresis Verification](#section12)
* [Comparison of Enzyme Activity](#section13)
* [Enzymatic Properties Measurement](#section14)
* [Determination of Kinetic Parameters](#section15)
* [High Performance Liquid Chromatography (HPLC) Assay](#section16)

Preparation of LB Medium

Table 1 LB medium formula

|  |  |
| --- | --- |
| Tryptone | 10g/L |
| NaCl | 10g/L |
| Yeast | 5g/L |

Construction of Soluble Expression Module Plasmids

The fusion tags NusA, TrxA, and MBP were individually cloned from plasmids previously stored in the laboratory. The AJC7 gene was amplified from a pET-28a(+)-AJC7 plasmid, which had been synthesized by a commercial provider.

Table 2 Primers used in the experiments

|  |  |
| --- | --- |
| Primers | Sequence |
| AJC7-Rev | GCCGCTGCTGTGATGATGATGATG |
| AJC7-For | ATGTTCGCAGAATTTCAGCATCTG |
| Crbq-Rev | CAGATGCTGAAATTCTGCGAACATACTGCCACCGCCACCGCTACCG |
| MBP-For | CATCATCATCATCACAGCAGCGGCARGAAAATCGAAGAAGGTAAAC |
| NusA-For | CATCATCATCATCACAGCAGCGGCAACAAAGAAATTTTGGCTGTAG |
| TrxA-For | CATCATCATCATCACAGCAGCGGCAGCGATAAAATTATTCACCTG |

Table 3 reaction syste

|  |  |
| --- | --- |
| Component | Volume |
| ddH2O | Up to 50 µL |
| 2 × Phanta SE Buffer | 25 µL |
| Primer F (10 μM ) | 2 µL |
| Primer R(10 μM ) | 2 µL |
| Phanta SE Super-Fidelity DNA Polymerase | 1 µL |
| Template DNA | x µL |

Linearization of pET-28a(+)-AJC7 Vectors

Table 4 reaction system

|  |  |
| --- | --- |
| Component | Volume |
| ddH2O | Up to 50 µL |
| 2 × Phanta SE Buffer | 25 µL |
| Primer F (10 μM ) | 2 µL |
| Primer R(10 μM ) | 2 µL |
| Phanta SE Super-Fidelity DNA Polymerase | 1 µL |
| Template DNA | x µL |

Table 5 Standard reaction procedure

|  |  |  |  |
| --- | --- | --- | --- |
| Cycle steps | Temperature | Time | Number of Cycles |
| pre-denaturation | 98℃ | 30 sec |  |
| denaturation | 98℃ | 10 sec | 28-35cycles |
| annealing | Tm | 5 sec | 28-35cycles |
| extension | 72℃ | 5 - 10 sec/kb | 28-35cycles |
| Complete extension | 72℃ | 1 min |  |

Gibson Assembly and Heat Shock Transformation

1. Gibson Assembly:The appropriate DNA fragments and vector were mixed, and ddH2O was added to adjust the final volume to 5 µL.5 µL of 2× CE Mix was then added.The reaction was carried out using Vazyme’s ClonExpress Ultra One Step Cloning Kit V2.

2. Heat Shock Transformation:

a. Remove competent BL21 (DE3) cells from the -80°C freezer and place them on ice.

b. Once thawed (10 minutes), 10 µL of plasmid was added to 100 µL of competent cells, followed by incubation on ice for 30 minutes.

c. Heat shock the cells at 42°C for 90 seconds, then immediately return them to ice for 2–3 minutes.

d. Add 700 µL of antibiotic-free LB medium and incubate at 37°C on a shaker for 1 hour.

e. Plate 100 µL of the transformed cells onto LB agar plates containing the appropriate antibiotics.

f. Invert the plates and incubate them overnight at 37°C in a constant temperature incubator.

3. Screening and Colony Verification:After heat shock transformation, LB plates containing Kanamycin were used for initial screening.Inverted plates were incubated at 37°C for 12 hours to allow for colony growth, which was subsequently used for verification.

Dual Plasmid Transformation

1. Preparation of Plasmid Mixture:Equal amounts of the molecular chaperone plasmid and the pET-28a(+)-AJC7 plasmid were mixed.

2. Heat Shock Transformation:

a. Remove competent BL21 (DE3) cells from the -80°C freezer and place them on ice.

b. After thawing (10 minutes), 10 µL of the plasmid mixture was added to 100 µL of competent cells, followed by incubation on ice for 30 minutes.

c. Heat shock the cells at 42°C for 90 seconds, then immediately place them back on ice for 2 minutes.

e. Plate 100 µL of the transformed cells onto LB agar plates containing the appropriate antibiotics.

f. Invert the plates and incubate overnight at 37°C in a constant temperature incubator.

3. Screening and Colony Selection:After heat shock transformation, LB plates containing Kanamycin (Kan) and Chloramphenicol (Cm) were used for initial screening.The plates were inverted and incubated at 37°C for 12 hours to allow for colony growth and subsequent verification.

Product Purification

The 10× DNA loading buffer was mixed with the PCR amplification product at a 1:10 ratio and gently mixed. The mixture was subjected to 1% agarose gel electrophoresis in 1× TAE buffer, running at 105 V for approximately 30 minutes. The gel was then examined under UV light to verify that the bands corresponded to the target fragment size.

Target fragments were recovered using the FastPure R Gel DNA Extraction Mini Kit (Nanjing Novogene Bioinformatics Technology Co. Ltd.), following the detailed protocol provided. The purified DNA products were stored at -20°C for future use.

Construction Of Mutation Module Plasmids

Table 6. Primers used in the experiments

|  |  |
| --- | --- |
| Primer | Sequence |
| AJC7-S125D-F | GCACAGCAGGATGTTCGCGAAATTAGTCGTACC |
| AJC7-S125D-R | TTCGCGAACATCCTGCTGTGCAAAAATCGG |
| AJCT-I129T-F | GTTCGCGAAACCAGTCGTACCGAACGTAATTG |
| AJCT-I129T-R | GGTACGACTGGTTTCGCGAACACTCTGC |
| AJCT-L140P-F | CTGGATGTGCCGCATAGTGCCGTTTGGGG |
| AJCT-L140P-R | GGCACTATGCGGCACATCCAGCCAATTACGT |
| AJC7-T181A-F | ACCATGTTTGCTATTGATCCGAGTGATCATGTTAA |
| AJC7-T181A-R | CGGATCAATAGCAAACATGGTATAACCGGCAC |
| AJC7-H342L-F | CTGAGCCTGCTTAGTGGTAGTGATAAATTTTCTGTTTACCC |
| AJC7-H342L-R | ACTACCACTAAGCAGGCTCAGGCGATAAC |

Table 7 reaction system

|  |  |
| --- | --- |
| Competent | Volume |
| ddH2O | Up to 50 µL |
| 2 × Phanta SE Buffer | 25 µL |
| Primer F (10 μM ) | 2 µL |
| Primer R (10 μM ) | 2 µL |
| Phanta SE Super-Fidelity DNA Polymerase | 1 µL |
| Template DNA | x µL |

Table 8 special reaction procedures

|  |  |  |  |
| --- | --- | --- | --- |
| Cycle steps | Temperature | Time | Number of Cycle |
| pre-denaturation | 98℃ | 30 sec |  |
| denaturation | 98℃ | 10 sec | 28-35cycles |
| annealing | Tm | 5 sec | 28-35cycles |
| extension | 72℃ | 5 - 10 sec/kb |  |
| Complete extension | 72℃ | 1 min |  |

At the end of the PCR reaction, 1 µL of DpnI and 5 µL of 10× EC Buffer were added to the reaction mixture for demethylation. The mixture was incubated at 37°C for 40 minutes, followed by a heat inactivation step at 80°C for 20 minutes. After digestion, 5 µL of the reaction was taken for verification by nucleic acid gel electrophoresis.

Colony PCR and Sequencing

For PCR validation of the fusion tag colonies, the following primers and systems were used:AJC7-Rev and AJC7-For were utilized to verify the presence of the AJC7 target gene on the plasmid.MBP-For, NusA-For, TrxA-For, and Crbq-Rev were employed to confirm the successful attachment of the fusion tags to the plasmid.

Table 9 Primers used in the experiments

|  |  |
| --- | --- |
| Primer | Sequence |
| AJC7-Rev | GCCGCTGCTGTGATGATGATGATG |
| AJC7-For | ATGTTCGCAGAATTTCAGCATCTG |
| Crbq-Rev | CAGATGCTGAAATTCTGCGAACATACTGCCACCGCCACCGCTACCG |
| MBP-For | CATCATCATCATCACAGCAGCGGCARGAAAATCGAAGAAGGTAAAC |
| NusA-For | CATCATCATCATCACAGCAGCGGCAACAAAGAAATTTTGGCTGTAG |
| TrxA-For | CATCATCATCATCACAGCAGCGGCAGCGATAAAATTATTCACCTG |

Table 10 Colony PCR reaction system

|  |  |
| --- | --- |
| Component | Volume |
| 2×Rapid Taq Master Mix | 5 µL |
| Primer F(100µM) | 0.4 µL |
| Primer R(100µM) | 0.4 µL |
| bacterial colony | not include |
| ddH2O | Up to 10 µL |

1.The PCR reaction solution was prepared according to the system described above.

2.Multiple single colonies were picked from the petri dishes cultured overnight after transformation, and each was introduced into the PCR reaction system using sterile tips.

3.After the PCR reaction was completed, nucleic acid gel electrophoresis was used to verify the presence of the desired fragment. If a fragment of the expected size was observed, the corresponding colony was streaked onto a fresh plate and subsequently transferred to a liquid medium for growth.

4.The strains were preserved by mixing the bacterial culture with glycerol at a 1:1 ratio. Remaining samples were prepared and sent for sequencing.

Cell Cultivation and Induction

1.After successful sequencing, the retained strains were inoculated into a shake tube containing 5 mL of LB (Kan) liquid medium and incubated at 37°C with shaking for approximately 5 hours.

2.Subsequently, 2 mL of the bacterial culture was transferred to a flask containing 50 mL of LB (Kan) medium.

3.The culture was shaken at 200 rpm at 37°C until the OD600 reached 0.6.

4.IPTG was then added to the culture to a final concentration of 0.5 mM in a sterile environment.

5.The culture was incubated at 20°C with shaking at 200 rpm for 16 hours.

Cell Fragmentation and Protein Harvesting

1.Microorganism Collection:After induction, the bacterial culture was centrifuged at 8000 rpm for 5 minutes, and the supernatant was discarded. The bacterial pellet was resuspended in 5 mL of PBS to wash away any residual medium. The suspension was centrifuged again at 8000 rpm for 5 minutes, the supernatant was discarded, and the pellet was resuspended in 3 mL of PBS.

2.Cell Disruption:The resuspended cells were placed in an ultrasonic crusher set at approximately 300 W. High-frequency ultrasonic vibrations were applied in cycles of 3 seconds on, followed by a 5-second interval, for a total of 10 minutes, until the bacterial solution became visibly clarified.

3.Protein Harvesting:Following ultrasonic disruption, the solution was centrifuged at 4°C at 12,000 rpm for 30 minutes to separate the supernatant (containing soluble proteins) from the pellet.

Protein Purification

1.Nickel Column Preparation:The nickel affinity column (stored in alcohol at -20°C) was prepared by first removing the alcohol. The column was then sequentially washed with 5 mL of Buffer A, followed by 3 mL of Buffer B, and finally another 5 mL of Buffer A.

2.Protein Binding and Washing:The crude enzyme solution was loaded onto the prepared nickel column. The column was washed twice with 5 mL of Buffer A to remove any impurity proteins. Subsequently, 3 mL of Buffer B was added, and the eluate was collected in a pre-marked beaker to obtain the target protein.

3.Column Maintenance:After collecting the target protein, another 3 mL of Buffer B was passed through the column. The column was then washed with 5 mL of Buffer A and stored in 20% alcohol for future use.

4.Protein Concentration via Ultrafiltration:The crude enzyme solution collected in the beaker was transferred into an ultrafiltration tube, which was sealed with ultrapure water. The tube was centrifuged at 4°C, 6500 rpm for 30 minutes.

5.Buffer Exchange:After centrifugation, 2 mL of ultrapure water was added to the ultrafiltration tube, and centrifugation was repeated at 6500 rpm for another 30 minutes.

6.Final Concentration:Finally, an additional 2 mL of ultrapure water was added, and the solution was centrifuged at 6500 rpm for 15 minutes. If the volume of the purified enzyme solution exceeded 500-700 µL, further centrifugation (for an additional 5-15 minutes) was performed to increase the protein concentration.

7.Storage:The concentrated purified enzyme solution was stored at -20°C for future use.

SDS-PAGE Electrophoresis Verification

Following the SDS-PAGE protocol detailed in [protocol], the sample preparation was done based on the protein concentration. The supernatant, pellet, and purified enzyme solution were then subjected to SDS-PAGE electrophoresis to determine if a band corresponding to the target protein was present.

Comparison of Enzyme Activity

The 500 µL reaction system consisted of the following components: purified enzyme solution at a final concentration of 1 mg/mL, D-fructose at a final concentration of 10 g/L, and 1 mM of the metal catalytic ion Ni2+. The required volumes of enzyme solution and substrate were added, and the final volume was brought to 500 µL with 50 mM Tris-HCl buffer (pH 9.0). The reaction was conducted at 70°C for 5 hours.

After the reaction, 5 µL of 1% H2SO4 was added to stop the reaction. The enzyme was then inactivated by heating at 100°C for 10 minutes. Following centrifugation, the supernatant was filtered through a 0.22 µm pore size membrane and analyzed using HPLC.

Enzymatic Properties Measurement

1.Optimum pH of the Reaction:
The enzyme solution was incubated in a 50 mM buffer solution (pH 3–11) with a substrate concentration of 100 g/L D-fructose and a final enzyme concentration of 0.1 mg/mL for 3 hours. The following buffer systems were used:pH 3–5: Citric acid-sodium citrate;pH 6–7: Na2HPO4-NaH2PO4;pH 8–9: Tris-HCl;pH 10–11: NaOH-NaH2PO4

2.Optimum Reaction Temperature:
The reaction mixture contained 100 g/L D-fructose in the buffer, a final enzyme concentration of 0.1 mg/mL, and 1 mM Ni2+, with the remaining volume brought to 500 µL using Tris-HCl buffer (pH 9.0). The reaction was performed at five different temperatures: 50°C, 60°C, 70°C, 80°C, and 90°C, each for 2 hours.

3.Maximum Conversion Rate of the Reaction:
The reaction system contained 100 g/L D-fructose, a final enzyme concentration of 23 mg/mL, and 1 mM Ni2+, with the total volume adjusted to 500 µL using Tris-HCl buffer (pH 9.0). The reaction was carried out at 70°C in a metal oscillator. Samples (40 µL) were collected every 20 minutes for a total of 180 minutes. To stop the reaction, 1 µL of 1% H2SO4 was added to each sample, which was then diluted to 200 µL using Tris-HCl buffer (pH 9.0).

Determination of Kinetic Parameters

The substrate concentration gradient for D-fructose was set as follows: 5 g/L, 10 g/L, 20 g/L, 40 g/L, 50 g/L, 60 g/L, 80 g/L, and 100 g/L. The final enzyme concentration in the reaction system was 5 mg/mL, with 1 mM Ni2+ as the catalytic metal ion. The remaining volume was adjusted with 50 mM Tris-HCl buffer (pH 9.0). The reaction was performed at 70°C for 20 minutes on a metal oscillator. The concentration of tagatose in the samples was determined using HPLC.

High Performance Liquid Chromatography (HPLC) Assay

The HPLC analysis was performed using an Ultimate® XB-NH2 column (4.6 × 250 mm, 5 µm).

Table 11 Chromatographic Conditions:

|  |  |
| --- | --- |
| Mobile phase | 75 % acetonitrile solution |
| Flow velocity Sample size | 1.0mL/min 10μL |
| Column temperature | 30℃ |
| Detector | RID |
| Detection wavelength | RID detection temperature：35℃ |

Preparation of the Mobile Phase:
Combine 750 mL of chromatographically pure acetonitrile with 250 mL of ultrapure water. Mix thoroughly and degas the solution using ultrasonic treatment to ensure homogeneity.

Preparation of Mixed Reference Solution:
Weigh approximately 1 mg each of D-fructose and D-tagatose, and dissolve them in 300 µL of ultrapure water in the same injection vial. Then, add 700 µL of chromatographic-grade pure acetonitrile and shake the vial to ensure complete mixing.

About Us：

Contact:

Address: 30 South Puzhu Road, Nanjing 211816, Jiangsu Province, China

![](https://static.igem.wiki/teams/5366/footer/f1.jpg)
![](https://static.igem.wiki/teams/5366/footer/f2.jpg)

![](https://static.igem.wiki/teams/5366/footer/f4.jpg)

---

© 2024 - Content on this site is licensed under  [Creative Commons Attribution 4.0 International license.](https://creativecommons.org/licenses/by/4.0/)

The repository used to create this website is available at [gitlab.igem.org/2024/njtech-china-a.](https://2024.igem.wiki/njtech-china-a/)

![](https://static.igem.wiki/teams/5366/home/home-picture/band.png)
[NJTech-China-A](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27home%27%29%20%7D%7D)

* [Project](#option1)

  [Description](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27description%27%29%20%7D%7D)
  [Contribution](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27contribution%27%29%20%7D%7D)
  [Protocol](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27protocol%27%29%20%7D%7D)
  [Engineering](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27engineering%27%29%20%7D%7D)
  [Experiments](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27experiments%27%29%20%7D%7D)
  [Notebook](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27notebook%27%29%20%7D%7D)
  [Results](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27results%27%29%20%7D%7D)
* [Part](#option2)

  [Part](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27part%27%29%20%7D%7D)
* [Model](#option3)

  [Model](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27model%27%29%20%7D%7D)
* [Team](#option4)

  [Members](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27members%27%29%20%7D%7D)
  [Attribution](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27attributions%27%29%20%7D%7D)
* [Human Practices](#option5)

  [Human Practice](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27human-practices%27%29%20%7D%7D)
  [Education](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27education%27%29%20%7D%7D)
  [Communication&Collaboration](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27communication%27%29%20%7D%7D)

# Results

* [A. New Enzyme Mining of Tagatose 4-Epimerase](#section1)
* [B. Fusion-promoting Tags](#section2)
* [C. Molecular chaperones](#section3)
* [D. Fermentation optimization](#section4)
* [E. Mutation](#section5)
* [F. Determination of Enzymatic Properties and Enzyme Kinetic Parameters](#section6)

Upon reviewing the iGEM part library, we discovered that past teams, such as XJTU-China 2018, had also provided sequences for enzymes capable of catalyzing the generation of tagatose from fructose. However, these teams did not present any experimental data. In contrast, our team proposes an enzyme that catalytically generates tagatose from fructose, supported by wet experimental data for the first time in iGEM history. Furthermore, we modified this enzyme through protein engineering, enhancing its conversion rate to 37%. While comparable data from previous iGEM entries is lacking, literature review indicates that our proposed enzyme remains competitive among others with similar functionalities.

A. New Enzyme Mining of Tagatose 4-Epimerase

The amino acid sequence UxaE, derived from *Thermotoga petrophila* RKU-1, served as a template for a BLAST search against the non-redundant protein sequence database of the National Center for Biotechnology Information (NCBI). The top 100 sequences exhibiting high sequence similarity were selected. Multiple sequence alignments were conducted using MEGA software, and after removing mismatched sequences, a phylogenetic tree was constructed using the adjacency method (Fig. 1).

![](https://static.igem.wiki/teams/5366/results/1.png)

Figure 1 The phylogenetic tree was constructed by BLAST search of the amino acid sequence UxaE derived from *Thermotoga petrophila* RKU-1 as a template

AlphaFold2 was utilized to model the structures of 80 sequences with a similarity threshold of less than 70% (Fig.2).

![](https://static.igem.wiki/teams/5366/results/2.png)

Figure 2 Alphafold2 structural modeling ( AJC7 as an example)

C-DOCKER, a molecular docking protocol based on the CHARMm force field, was employed in Discovery Studio to dock the ligand to the receptor binding site. The binding free energy of the acceptor-ligand complex was calculated using a CHARMm-based energy function and the implicit solvent method within Discovery Studio. The binding free energy between the selected sequence and fructose is illustrated in Fig. 3.

![](https://static.igem.wiki/teams/5366/results/03.png)

Figure 3 The binding free energy between the selected sequence and fructose

Considering the binding free energy as well as the branches and sources in the phylogenetic tree, we selected four potential proteins with tagatose-4-epimerase activity, naming them MBC, AJC7, TET, and HDM, respectively. These four enzymes were evaluated for their ability to catalyze the conversion of fructose to tagatose under identical conditions. The resulting tagatose was analyzed using high-performance liquid chromatography (HPLC), and the results are presented in Fig. 4. According to the HPLC data, the activity of wild-type AJC7 significantly surpassed that of wild-type UxaE, with tagatose concentration produced under the same catalytic conditions being approximately four times greater than that of UxaE. Given its promising tagatose-4-epimerase activity, AJC7 was selected for further research.

![](https://static.igem.wiki/teams/5366/results/4.png)

Figure 4 Concentrations of tagatose produced by wild-type UxaE, AJC7, HDM, MBC, and TET under the same reaction conditions

B. Fusion-promoting Tags

Plasmid design

To improve the wild-type enzyme activity of AJC7, we integrated three fusion-promoting tags with the AJC7 sequence using a GGGGSGGGGS linker. The resulting recombinant plasmids were denoted as pET-28a(+)-MBP-AJC7, pET-28a(+)-NusA-AJC7, and pET-28a(+)-TrxA-AJC7.

![](https://static.igem.wiki/teams/5366/results/5.jpg)

![](https://static.igem.wiki/teams/5366/results/6.jpg)

![](https://static.igem.wiki/teams/5366/results/7.jpg)

![](https://static.igem.wiki/teams/5366/results/8.jpg)

Figure 5 Plasmid maps of recombinant plasmids PET-28a(+)-MBP-AJC7, pET-28a(+)-NusA-AJC7, pET-28a(+)-TrxA-AJC7

The fusion-promoting tag fragment underwent PCR amplification, while the pET-28a(+)-AJC7 vector was linearized via reverse PCR. Gel electrophoresis was employed to recover the correct nucleic acid fragment. After homologously recombining the tag fragment with the vector, heat shock transformation was carried out. Colonies that grew on plates were selected, cultured, and subjected to colony PCR to confirm the presence of recombinant plasmids (Fig. 6). The sizes of the TrxA, NusA, and MBP fragments were 402 bp, 1560 bp, and 1179 bp, respectively. Validated colonies from colony PCR were sequenced to verify the correct construction of plasmids with fusion tags. The accurate sequences were utilized for inducible expression. The strains altered with fusion-promoting tags were named TrxA-AJC7, NusA-AJC7, and MBP-AJC7, respectively. Following expression induction, cell lysates were separated into supernatant and pellet fractions for SDS-polyacrylamide gel electrophoresis analysis.

![](https://static.igem.wiki/teams/5366/results/9.png)

Figure 6 Nucleic acid gel plot of colony PCR

![](https://static.igem.wiki/teams/5366/results/007.png)

![](https://static.igem.wiki/teams/5366/results/071.jpg)

Figure 7 SDS-PAGE of recombinant AJC7 with a fusion-promoting tag

M: protein marker; Lane 1: precipitation of AJC7; Lane 2: supernatant of AJC7; Lane 3: precipitation of TrxA-AJC7; Lane 4: supernatant of TrxA-AJC7; Lane 5: precipitation of NusA-AJC7; Lane 6: supernatant of NusA-AJC7; Lane 7: precipitation of MBP-AJC7; Lane 8: supernatant of MBP-AJC7. The molecular weights are as follows: AJC7 at 60.4 kDa, TrxA-AJC7 at 70.4 kDa, NusA-AJC7 at 113.4 kDa, and MBP-AJC7 at 99.0 kDa.

As shown in Fig. 7, there is little difference between the supernatant and pellet of the original AJC7 (lanes 1 and 2) in the cell lysate, indicating limited soluble expression of the target protein. The concentration of the 70.4 kDa protein band (lane 4) in the supernatant significantly increased, while the proportion of the precipitated protein band (lane 3) decreased. Similarly, the concentration of the 113.4 kDa protein band (lane 6) in the supernatant rose significantly, with a corresponding reduction in the precipitated band. The 99.0 kDa protein band (lane 8) also showed a significant increase in the supernatant, and the precipitated band appeared notably lighter. Induced at 20℃for 16 hours, further densitometry analysis of the protein gel was conducted to quantify the increase in protein expression, with the results detailed below:

![](https://static.igem.wiki/teams/5366/results/11.jpg)

Figure 8 Histogram of densitometry data for bacterial cell breakage before and after ablation tag attachment, SDS-PAGE gel supernatant, and precipitated protein bands

![](https://static.igem.wiki/teams/5366/results/12.jpg)

Figure 9 Percentage comparison of densitometry data of bacterial cell breakage, supernatant, and precipitated protein bands before and after thawing tag attachment

Fig. 9 and 10 demonstrate that, under specific induction conditions, the solubility of the protein was significantly enhanced by NusA, TrxA, and MBP, with increases of 6.86, 6.79, and 6.92 times, respectively, compared to AJC7. Additionally, the density values of the protein bands in the precipitate were greatly reduced. This provides further validation for the inference that the incorporation of the fusion tag effectively improves the solubility of the target protein.

C. Molecular chaperones

Molecular chaperones were utilized in this study. The chaperone plasmids pG-KJE8, pGro7, pKJE7, pG-Tf2, and pTf16 were co-expressed with the pET-28a(+)-AJC7 plasmid to facilitate correct folding of exogenous proteins in prokaryotic cells, thereby enhancing the solubility of these proteins. This approach aimed to yield a substantial quantity of soluble proteins for exploring their biological functions. Modified strain pairs were denoted as follows: pG-KJE8/AJC7, pGro7/AJC7, pKJE7/AJC7, pG-Tf2/AJC7, and pTf16/AJC7. The chaperone plasmids were introduced into *E. coli* BL21 (DE3) host cells alongside the pET-28a(+)-AJC7 plasmid via thermomechanical transformation and initially screened on Kanamycin and Chloramphenicol-resistant plates. Single colonies that tested positive through colony PCR were subsequently induced for protein expression and subjected to purification processes.Following induction, the cellular supernatant and precipitate were separately analyzed via SDS-polyacrylamide gel electrophoresis.

![](https://static.igem.wiki/teams/5366/results/13.png)

Figure 10 SDS-PAGE co-expressed by chaperone and pET-28a(+)-AJC7

M: protein marker; Lane 1: precipitation of AJC7; Lane 2: supernatant of AJC7; Lane 3: precipitation of pG-KJE8/AJC7; Lane 4: supernatant of pG-KJE8/AJC7; Lane 5: precipitation of pGro7/AJC7; Lane 6: supernatant of pGro7/AJC7; Lane 7: precipitation of pKJE7/AJC7; Lane 8: supernatant of pKJE7/AJC7; Lane 9: precipitation of pG-Tf2/AJC7; Lane 10: supernatant of pG-Tf2/AJC7; Lane 11: precipitation of pTf16/AJC7; Lane 12: Supernatant of pTf16/AJC7.

Further analysis of the protein gel was conducted using densitometry to quantify the increase in protein expression.

![](https://static.igem.wiki/teams/5366/results/14.jpg)

Figure 11 Histogram of densitometry analysis data of SDS-PAGE gel supernatant and precipitated protein bands of bacterial cell breakage fluid co-expressing the target gene and chaperone protein

![](https://static.igem.wiki/teams/5366/results/15.jpg)

Figure 12 Percentage comparison of densitometry data of bacterial cell breakage, supernatant and precipitated protein bands co-expressed with chaperone proteins

As shown in Fig.11 and 12, the protein solubility of pG-Tf2/AJC7 increased by 2.2 times compared to AJC7, and the density of the protein bands in the precipitate decreased. These results indicate that the addition of chaperones significantly enhances the solubility of the target protein.

D. Fermentation optimization

1. Optimal temperature

Recombinant *E. coli* BL21 (DE3) was cultivated at 37℃ with agitation at 200 rpm. Upon reaching an optical density of 0.6 at 600 nm, 0.5 mM isopropylthiogalactoside (IPTG) was added, followed by incubation at 16℃, 20℃, 30℃, and 37℃ for 16 hours with agitation at 200 rpm to induce enzyme expression.

![](https://static.igem.wiki/teams/5366/results/16.png)

Figure 13 SDS-PAGE of recombinant AJC7 induced at different temperatures

M: marker; Lane 1: precipitation of enzyme AJC7 after induction at 16℃; Lane 2: precipitation of enzyme AJC7 after induction at 20℃; Lane 3: precipitation of enzyme AJC7 after induction at 30℃; Lane 4: precipitation of enzyme AJC7 after induction at 37℃; Lane 5:supernatant of enzyme AJC7 after induction at 16℃; Lane 6: supernatant of enzyme AJC7 after induction at 20℃; Lane 7: supernatant of enzyme AJC7 after induction at 30℃; Lane 8: supernatant of enzyme AJC7 after induction at 37℃.

Induced at varying temperatures for 16 hours, further densitometry analysis of the protein gel was performed to quantify the increase in protein expression, with the results detailed below:

![](https://static.igem.wiki/teams/5366/results/17.jpg)

Figure 14 Histogram of densitometry analysis data of SDS-PAGE gel supernatant and precipitated protein bands of bacterial cell breakage fluid after induction at different temperatures

![](https://static.igem.wiki/teams/5366/results/18.jpg)

Figure 15 Comparison of optical densitometry data percentages of bacterial cell breakage, supernatant and precipitated protein bands after different temperature induction

The outcomes are depicted in Fig.14 and 15. These figures illustrate a 1.24-fold elevation in the soluble expression of AJC7 at 20°C compared to induction at 16°C. Moreover, the intensity of protein bands in the precipitate significantly decreased, reinforcing the notion that selecting the appropriate induction temperature effectively enhances the target protein's solubility.

2. Optimal IPTG Concentration

Recombinant *E. coli* BL21 (DE3) was cultured at 37℃ with agitation at 200 rpm. Upon the culture's optical density reaching 0.6 at 600 nm, final concentrations of 0.1 mM, 0.2 mM, 0.5 mM, and 1 mM isopropylthiogalactoside (IPTG) were added successively. Subsequently, the culture was incubated at 20℃ for 16 hours with agitation at 200 rpm to induce enzyme expression.

![](https://static.igem.wiki/teams/5366/results/19.png)

Figure 16 SDS-PAGE of recombinant AJC7 induced by different concentrations of IPTG

M: protein marker; Lane 1: precipitation of enzyme AJC7 after 0.1 mM induction; Lane 2: precipitation of enzyme AJC7 after 0.2 mM induction; Lane 3: precipitation of enzyme AJC7 after 0.5 mM induction; Lane 4: precipitation of enzyme AJC7 after 1 mM induction; Lane 5: supernatant of enzyme AJC7 after 0.1 mM induction; Lane 6: supernatant of enzyme AJC7 after 0.2 mM induction; Lane 7: supernatant of enzyme AJC7 after 0.5 mM induction; Lane 8: supernatant of enzyme AJC7 after 1 mM induction.

At 20℃, AJC7 was induced with different concentrations of IPTG for 16 hours. Further densitometry analysis of the protein gel was performed to quantify the increase in protein expression, with the results detailed below:

![](https://static.igem.wiki/teams/5366/results/20.jpg)

Figure 17 Histogram of densitometry data of SDS-PAGE gel supernatant and precipitated protein bands of bacterial cell breakage fluid induced by different concentrations of IPTG

![](https://static.igem.wiki/teams/5366/results/21.jpg)

Figure 18 Percentage comparison of densitometry data of bacterial cell breakage, supernatant and precipitated protein bands after IPTG induction at different concentrations

The results, shown in Fig.17 and 18, revealed a 1.39-fold, 1.43-fold, and 1.48-fold enhancement in protein solubility after induction with 0.2 mM, 0.5 mM, and 1 mM IPTG, respectively. Additionally, a substantial decrease in the density of protein bands in the precipitate was observed.

3.Self-inducing medium

An orthogonal experimental design was employed to create sixteen groups of self-inducing media with varying carbon source combinations. Recombinant *E. coli* BL21 (DE3) was initially inoculated at 37℃ for 3 hours with agitation at 200 rpm, followed by incubation at 20℃, and 200 rpm for 20 hours to induce enzyme expression. Upon reaching an optical density of 0.6 at 600 nm in standard LB medium, a final concentration of 0.1 mM IPTG was introduced for induction control purposes.

Subsequent to induction at 20℃ for 20 hours, densitometry analysis of the protein gel was conducted to quantify the upsurge in protein expression, with the results scrutinized as outlined below:

A

![](https://static.igem.wiki/teams/5366/results/22.png)

B

![](https://static.igem.wiki/teams/5366/results/23.png)

C

![](https://static.igem.wiki/teams/5366/results/24.png)

Figure 19 SDS-PAGE of recombinant AJC7 after induction in autoinduction medium with different combinations of carbon sources

M: protein marker; Lane C: supernatant of enzyme AJC7 after induction in the normal LB control group; Lanes 1-16: supernatant of enzyme AJC7 after induction in experimental groups 1-16; Lane C': precipitation of enzyme AJC7 after induction in the normal LB control group; Lanes 1'-16': precipitation of enzyme AJC7 after induction in experimental groups 1-16.

Induced at 20℃ for 20 hours, further densitometry analysis of the protein gel was performed to quantify the increase in protein expression, with the results detailed below:

![](https://static.igem.wiki/teams/5366/results/25.jpg)

Figure 20 Histogram of densitometry data of bacterial cell breakage fluid SDS-PAGE gel supernatant and precipitated protein bands after induction in autoinduction medium with different combinations of carbon sources

![](https://static.igem.wiki/teams/5366/results/26.png)

Figure 21 Comparison of optical density analysis data of bacterial cell breakage, supernatant, and precipitated protein bands after induction in autoinduction medium with different combinations of carbon sources

Fig.20 and 21 indicate that the solubility of the target protein increased by 1.82 times, 2.33 times, 2.15 times, 2.36 times, and 2.15 times in experimental groups 1, 2, 14, 15, and 16, respectively, compared to the standard LB medium. Additionally, the density values of the protein bands in the precipitate were significantly reduced.

E. Mutation

1. Point mutations were introduced using designed primers for S125D, T181A, H342L, I129T, and L140P on the pET-28a(+)-AJC7 template to obtain single-point mutants (Fig.22). The PCR reaction system was followed by DpnI-mediated demethylation, nucleic acid gel electrophoresis verification, and subsequent recovery of PCR products. The concentration of each single-point mutant plasmid was measured before transfer to *E. coli* BL21 (DE3) and incubation at 37℃ for 14 hours invertedly. Individual colonies were selected post-transformation, subjected to colony PCR, and validated using nucleic acid gel electrophoresis as presented in Figure 23. After validation, colonies with correct bands were transferred to LB (Kan) liquid medium, and sequencing confirmed successful construction of all five single-point mutant plasmids.

2. The three distantly located mutation points, T181A, H342L, and L140P, were used with the template pET-28a(+)-AJC7-S125D to construct S125D/T181A, S125D/H342L, and S125D/L140P two-point mutants. Given the proximity of I129T to S125D, primers from the I129T spike served as the template for constructing the S125D/I129T mutant.

3. Three-point mutants, S125D/T181A/I129T, S125D/T181A/L140P, and S125D/T181A/H342L, were successfully developed using the method detailed in [engineering].

4.The four-point mutant, S125D/T181A/I129T/L140P, and the five-point mutant, S125D/T181A/I129T/L140P/H342L, of AJC7 were successfully created with the method outlined in [engineering].

![](https://static.igem.wiki/teams/5366/results/27.png)

Figure 22 Point mutation localization and primer design

![](https://static.igem.wiki/teams/5366/results/28.jpg)

Figure 23 Nucleic acid gel diagram of PCR with two-point mutant colonies

![](https://static.igem.wiki/teams/5366/results/29.jpg)

Figure 24 Nucleic acid gel diagram of PCR with two-point mutant colonies

![](https://static.igem.wiki/teams/5366/results/30.jpg)

Figure 25 Nucleic acid gel diagram of PCR of three-point mutant colonies

![](https://static.igem.wiki/teams/5366/results/31.jpg)

Figure 26 Nucleic acid gel map of colony PCR (four-point mutation on the left and five-point mutation on the right)

Single-point mutations

Mutant and wild-type strains were activated, amplified, and cultured, followed by a series of protein purification operations to extract the protein of interest using the method outlined in [Experiment]. The volume of purified enzyme solution required for the 500 µL reaction system was determined based on the protein concentration, as shown in [Experiment]. The final concentration of fructose in the system was set at 100 g/L, with 10 µL of Ni2+ used as a catalyst. The reaction was conducted at 70℃ for 5 hours, and the product was analyzed using high-performance liquid chromatography (HPLC) (Fig.27).

![](https://static.igem.wiki/teams/5366/results/32.png)

Figure 27 The concentrations of tagose produced in the system after WT, S125D, T181A, H342L, I129T, and L140P reacted with 100 g/L substrate fructose for 5 h

The results indicated that the concentrations of AJC7 after five single-point mutations—S125D, T181A, H342L, I129T, and L140P—were higher than those of wild-type AJC7. The enhancements observed with the H342L, I129T, and L140P mutants were not significant. Notably, the catalytic efficiency of the S125D mutant was twice that of the wild type under identical reaction conditions and substrate concentrations.

Two point mutations

![](https://static.igem.wiki/teams/5366/results/33.jpg)

Figure 28 The concentrations of tagatose in wild-type, S125D, S125D/T181A, S125D/H342L, S125D/I129T, and S125D/L140P after reaction with 100g/L fructose substrate for 5 h, respectively

The results showed that the concentrations of AJC7 after the S125D/T181A, S125D/H342L, S125D/I129T, and S125D/L140P mutations were higher than those of both wild-type AJC7 and S125D. Notably, the S125D/T181A two-point mutant exhibited a significant increase in AJC7 enzyme activity, with its catalytic efficiency nearly twice that of wild-type AJC7.

Three-point mutation.

![](https://static.igem.wiki/teams/5366/results/34.png)

Figure 29 The concentrations of tagatose in wild-type, S125D, S125D/T181A, S125D/T181A/I129T, and S125D/T181A/H342L reacted with 100 g/L fructose substrate for 5 h, respectively

The results indicated that the concentrations of AJC7 after the S125D/T181A/I129T and S125D/T181A/H342L three-point mutations were higher than those of wild-type AJC7, S125D, and S125D/T181A. However, the S125D/T181A/L140P three-point mutant did not significantly enhance enzyme activity. Among the mutants, the S125D/T181A/I129T three-point variant demonstrated the greatest increase in AJC7 enzyme activity, with its catalytic efficiency nearly three times that of wild-type AJC7.Four-point.

Five-point mutation.

![](https://static.igem.wiki/teams/5366/results/35.png)

Figure 30 The concentrations of tagosse in WT, S125D, S125D/T181A, S125D/T181A/I129T, S125D/T181A/I129T/L140P,S125D/T181A/I129T/L140P/H342L after reacting with 100g/L fructose substrate for 5 h

The results indicated that the S125D/T181A/I129T/L140P four-point mutant and the S125D/T181A/I129T/L140P/H342L five-point mutant of AJC7 showed improvements compared to wild-type AJC7. However, the enzyme activity of the S125D/T181A/I129T mutant was not satisfactory when compared to that of the S125D/T181A/I129T mutant.

F. Determination of Enzymatic Properties and Enzyme Kinetic Parameters

Enzymatic properties studies

a.Relative activity was calculated under various pH conditions considering maximum activity at pH 9.0.

![](https://static.igem.wiki/teams/5366/results/36.png)

Figure 31 The relative activities of AJC7-S125D/T181A/I129T at different pH.
The activity at pH 9.0 was set as 100%

Examining Fig.31 reveals that AJC7 shows minimal catalytic activity for fructose to tagatose conversion at pH 4.0-5.0. The enzyme's activity increases with rising pH (6.0–9.0), peaking at pH 9.0 and declining beyond pH 9.0 to 11.0. Thus, AJC7 is most effective for fructose to tagatose conversion under slightly alkaline conditions.

b.The activity at 70℃ was set as 100%, with relative activity calculated for other temperature ranges.

![](https://static.igem.wiki/teams/5366/results/37.png)

Figure 32 AJC7-S125D/T181A/I129T triple mutant enzyme activity as a function of reaction temperature

Analysis of Fig.32 indicates that, at the optimum pH of 9.0, the enzyme activity of the AJC7-S125D/T181A/I129T three-point mutant rises gradually within the 50–70℃ range, reaching a peak at 70℃. Further increase in temperature leads to a decline in enzyme activity. Therefore, the optimal temperature for catalyzing fructose to tagatose conversion by the AJC7-S125D/T181A/I129T mutant is 70℃.

c. To determine the conversion rate of tagatose-4-epimerase AJC7, the substrate conversion rate of the optimal mutant was evaluated under optimal reaction conditions (70℃, pH 9.0, 1 mmol/L Ni2+).

![](https://static.igem.wiki/teams/5366/results/38.png)

Figure 33 Conversion rate of the best AJC7 mutant under optimal conditions

With an enzyme concentration of 23 mg/mL, the optimal mutant catalyzed the conversion of 100 g/L fructose to yield 37 g/L tagatose in 120 minutes, achieving a 37% conversion rate and a yield of 18.5 g/(L·h). At 80 minutes, 33 g/L tagatose was produced, translating to a 33% conversion rate and a yield of 24.1 g/(L·h), the highest recorded.

Enzyme kinetic parameter determination

![](https://static.igem.wiki/teams/5366/results/39.png)

Figure 34 Nonlinear regression equations for substrate concentration and reaction rate

In determining the Michaelis constant of the optimal mutant S125D/T181A/I129T of AJC7, a fructose concentration gradient was set (5g/L, 10g/L, 20g/L, 40g/L, 50g/L, 60g/L, 80g/L, 100g/L) with a final pure enzyme concentration of 0.1 mg/mL in the presence of 1mM Ni2+ catalytic metal ion and Tris-HCl buffer at 50mM pH=8.0. The reaction was conducted at 70℃ in a metal shaker for 20 minutes. Post-reaction, the yield of tagatose and residual fructose was determined by high-performance liquid chromatography to calculate the conversion rate. Using Origin software nonlinear regression, Km was calculated as 99.805 mM and Vmax as 27.309 mM/h, indicating the strong substrate affinity of the AJC7 S125D/T181A/I129T mutant.

About Us：

Contact:

Address: 30 South Puzhu Road, Nanjing 211816, Jiangsu Province, China

![](https://static.igem.wiki/teams/5366/footer/f1.jpg)
![](https://static.igem.wiki/teams/5366/footer/f2.jpg)

![](https://static.igem.wiki/teams/5366/footer/f4.jpg)

---

© 2024 - Content on this site is licensed under  [Creative Commons Attribution 4.0 International license.](https://creativecommons.org/licenses/by/4.0/)

The repository used to create this website is available at [gitlab.igem.org/2024/njtech-china-a.](https://2024.igem.wiki/njtech-china-a/)