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.
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.
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.
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.
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.
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.
Proteins were extracted and purified for tagatose production. The samples were analyzed using high-performance liquid chromatography (HPLC).
Based on the tagatose titer analyzed by HPLC, the best wild-type enzyme selected was AJC7.
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.
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.
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.
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.
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.
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.
Single colonies confirmed by colony PCR were activated in LB media with Cm and Kan. Colony PCR results were verified for correct re-transformation.
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 |
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.
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.
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.
The corresponding bands were quickly excised under UV light for gel recovery using the Novizen Product Purification Kit.
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.
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.
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.
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.
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.
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.
The HPLC results indicated that the AJC7-S125D mutant strain exhibited the highest enzyme activity among the tested samples.
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.
Two-point mutants, specifically S125D/T181A, S125D/H342L, S125D/I129T, and S125D/L140P, were engineered for further study.
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.
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.
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.
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.
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.
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.
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.
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.
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℃.
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.
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.
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.
No cells grew on the plates after the overnight culture.
The transformation was repeated and grown overnight at 37℃.
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.
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.
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.
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.
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.
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.
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.
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.
Repeat the 8.8-8.9 experiments, and still no green fluorescence was detected.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.