Notebook

Day 1 – 20240714

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Search and Expression Analysis of Key Gene Sequences

During the synthesis of VB6, PDXA and PDXJ are crucial, as they are the two key enzymes for in vitro synthesis of VB6. We obtained the DNA sequences of these two key genes through NCBI and previous literature reports.

PDXA gene

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PdxA gene sequence:

ATGGTTAAAACCCAACGTGTTGTGATCACTCCCGGCGAGCCCGCCGGGATTGGCCCGGACTTAGTTGTCCAGCTTGCACAGCGTGAGTGGCCGGTCGAACTGGTTGTTTGTGCCGATGCCACTCTCCTTACCAACCGGGCAGCGATGCTCGGTTTGCCGCTCACCCTCCGCCCTTATTCCCCCAACTCCCCTGCACAACCGCAAACTGCGGGCACATTAACGCTACTTCCTGTCGCGCTACGTGCACCTGTCACTGCGGGGCAGTTAGCGGTTGAAAATGGGCATTATGTGGTGGAAACGCTGGCGCGAGCGTGCGATGGTTGTCTGAACGGCGAATTTGCCGCGCTGATCACAGGTCCGGTGCATAAAGGCGTTATTAACGACGCTGGCATTCCTTTTACCGGTCATACCGAGTTTTTCGAAGAGCGTTCGCAGGCGAAAAAGGTGGTGATGATGCTGGCGACCGAAGAACTTCGCGTGGCGCTGGCAACGACGCATTTACCGCTGCGCGATATCGCAGACGCTATCACCCCTGCACTTTTGCACGAAGTGATTGCTATTTTGCATCACGATTTGCGGACCAAATTTGGTATTGCCGAACCGCGCATTCTGGTCTGCGGGCTGAATCCGCACGCGGGCGAAGGCGGTCATATGGGTACGGAAGAGATAGACACCATTATTCCGGTGCTCAATGAGCTGCGGGCGCAGGGGATGAAACTCAACGGGCCGCTGCCTGCCGATACCCTGTTTCAGCCGAAATATCTTGATAACGCCGACGCCGTGCTGGCGATGTACCACGATCAGGGTCTTCCCGTGCTAAAATACCAGGGCTTCGGGCGCGGTGTGAACATTACGCTGGGCCTGCCCTTTATTCGCACATCAGTGGACCACGGCACCGCGCTTGAACTGGCGGGACGTGGCAAAGCCGATGTCGGCAGTTTTATTACGGCGCTTAATCTCGCCATCAAAATGATTGTTAACACCCAATGA

We conducted codon optimization and analysis based on the above DNA sequence. The results are shown in the figure below:

PdxA Amino Acid Sequence:

MVKTQRVVITPGEPAGIGPDLVVQLAQREWPVELVVCADATLLTNRAAMLGLPLTLRPYSPNSPAQPQTAGTLTLLPVALRAPVTAGQLAVENGHYVVETLARACDGCLNGEFAALITGPVHKGVINDAGIPFTGHTEFFEERSQAKKVVMMLATEELRVALATTHLPLRDIADAITPALLHEVIAILHHDLRTKFGIAEPRILVCGLNPHAGEGGHMGTEEIDTIIPVLNELRAQGMKLNGPLPADTLFQPKYLDNADAVLAMYHDQGLPVLKYQGFGRGVNITLGLPFIRTSVDHGTALELAGRGKADVGSFITALNLAIKMIVNTQ*  

Amino acid sequence analysis revealed that there is no signal peptide or transmembrane region in this protein sequence, and it can be normally expressed.

Day 2 – 20240715

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PDXJ gene search and analysis

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PDXJ sequence:

ATGGCTGAATTACTGTTAGGCGTCAACATTGACCATATCGCTACGCTGCGCAACGCGCGCGGTACCGCTTACCCGGATCCGGTGCAGGCCGCGTTTATTGCCGAGCAGGCGGGAGCGGACGGCATTACCGTGCATTTACGTGAAGATCGCCGTCACATTACTGACCGCGACGTGCGCATCCTGCGTCAGACGCTGGATACCCGCATGAATCTGGAGATGGCGGTGACCGAAGAGATGCTGGCGATCGCCGTTGAGACGAAGCCACATTTTTGCTGCCTGGTACCGGAAAAGCGTCAGGAAGTAACAACCGAAGGCGGCCTGGATGTCGCAGGGCAGCGTGACAAAATGCGCGATGCCTGCAAACGTCTGGCAGATGCCGGGATTCAGGTTTCTCTGTTTATTGACGCCGATGAAGAGCAGATCAAAGCTGCGGCAGAGGTTGGCGCACCGTTTATCGAGATCCACACCGGTTGCTATGCTGATGCCAAAACTGACGCCGAACAGGCGCAAGAGCTGGCGCGTATCGCCAAAGCCGCGACCTTTGCCGCAAGCCTCGGTCTGAAAGTTAACGCCGGACACGGTCTGACCTATCACAACGTGAAAGCCATTGCCGCCATCCCTGAGATGCATGAACTGAATATCGGTCATGCCATTATTGGTCGTGCAGTGATGACCGGACTGAAAGATGCGGTGGCAGAAATGAAGCGTCTGATGCTGGAAGCGCGTGGCTAA

PDXJ Amino Acid Sequence:

MAELLLGVNIDHIATLRNARGTAYPDPVQAAFIAEQAGADGITVHLREDRRHITDRDVRILRQTLDTRMNLEMAVTEEMLAIAVETKPHFCCLVPEKRQEVTTEGGLDVAGQRDKMRDACKRLADAGIQVSLFIDADEEQIKAAAEVGAPFIEIHTGCYADAKTDAEQAQELARIAKAATFAASLGLKVNAGHGLTYHNVKAIAAIPEMHELNIGHAIIGRAVMTGLKDAVAEMKRLMLEARG*

Day 3 – 20240716

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Experiment Purpose:

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Synthesis of gene sequences

Experiment Process:

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Today’s main experiment was to verify the gene sequence on NCBI. After confirming the accuracy, the corresponding synthesis form was filled out and sent to the biological company for synthesis (to Synbio Technologies). Since the synthesis process takes a week, the next week will be a rest period, and subsequent experiments will begin once the sequence is received.

Day 4 – 20240723

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**Experiment Purpose: **

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Verification of PDXA and PDXJ amplification

Experiment Procedure:

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1. Turn on the PCR machine in advance.
2. Mix reagents on ice according to the table below in the PCR tubes:

3. Mix the solution system evenly. Carry out rapid centrifugation if the droplets hang on the tube wall.
4. Load the PCR tube into the PCR machine. Set the PCR program according to the table below.

Standard Agarose Gel Electrophoresis Procedure

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1. Preparation of the Gel Plate: Adjust the gel plate to level, place the gel tray, clamp it tightly, and insert the comb.
2. Weighing Agarose: Based on the gel plate’s volume and the required gel concentration, weigh the corresponding amount of agarose powder. For example, if the gel plate holds 20mL and a 1% gel (weight/volume concentration) is needed, weigh 0.2g of agarose powder.
3. Preparation of 1% Agarose Gel: Add 0.2g of agarose powder to 20mL of 0.5×TBE buffer, add 1μL GelRed, and mix well. Heat in the microwave until the solution becomes a clear, transparent liquid. Be careful not to boil the solution dry; take it out and check intermittently, heating in multiple short intervals if necessary.
4. Gel Formation: Carefully pour the prepared agarose solution into the gel plate. Allow it to spread evenly and ensure no bubbles form. Let it solidify at room temperature for 20–30 minutes.
5. Electrophoresis:
   1. After gently removing the comb, place the gel with the tray into the electrophoresis tank. Fill the tank with an appropriate amount of 0.5×TBE buffer, ensuring that the buffer just covers the gel and fills the sample wells (note: the wells should be near the negative electrode, black electrode).
   2. Loading Samples: Mix 10μL of DNA solution with 2μL of 6× loading buffer on hydrophobic wax paper (waxed paper). Carefully transfer the mixture into the sample wells using a pipette, allowing the DNA solution to sink slowly to the bottom of the wells. Leave one well empty (either on the sides or in the middle) and add 3μL of a DNA molecular weight marker (e.g., DL2000 Marker) to this well.
   3. Running the Electrophoresis: Close the lid of the electrophoresis tank (note: match the electrodes, black to black, red to red). Turn on the electrophoresis machine, select constant voltage, input the desired voltage, and, if needed, set a timer—then press start. Once the negative electrode generates a large number of bubbles, the electrophoresis has begun. After 30–40 minutes, the process should be ready for imaging. The exact time can be determined by checking the progress of the loading buffer (when it has migrated to about two-thirds of the gel). A voltage of 5V/cm is ideal—neither too high nor too low.
   4. Observation of Results: Once electrophoresis is complete, turn off the machine and remove the gel. Place the gel into the darkroom of the gel imaging system, turn on the imaging system, and open the control software on the computer. Use the software to control the camera for signal observation and photography. Adjust the focus, aperture, and image size as needed. Once the brightness and clarity are satisfactory, capture the image and save it.

Result

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Day 5 – 20240724

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Experiment Purpose:

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Construction of PDXA and PDXJ Expression Vectors

Procedure:

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1. Amplify the target gene. Sufficient amounts of PCR products were amplified by PCR.
2. The PCR products and plasmid vectors were digested separately according to the restriction sites designed by the primers.
3. Ligation of target fragments and vector: The digested PCR product was ligated to the plasmid vector using ligase.
4. The recombinant plasmids were then transformed into BL21(DE3) competent cells. Positive clones were screened and verified by sequencing.
5. Agarose Gel Electrophoresis Procedure: Refer to the previous manual.

Experimental Results:

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The pRSFDuet-1-PdxA-PdxJ expression plasmid was constructed. The synthetic sequence was first digested with enzymes to obtain specific enzyme-cutting ligation sites, and the ligation product was transformed into E. coli BL21(DE3) competent cells. Screening was carried out using ampicillin, resulting in a certain number of transformants. The plasmid was verified through enzyme digestion. As shown in the figure below, the size of the digested target gene fragment met the expected requirements.

Day 6 – 20240725

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Experimental Purpose:

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Transformation of the plasmid into E. coli.

Procedure:

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1. Remove the EPI400 competent cells from storage and place them on ice, ensuring the icebox temperature is below 0°C to thaw the EPI400 competent cells.
2. Transfer 100 μl of EPI400 competent cells into 10 μl of plasmid sample, flick to mix well (avoid touching the competent cells with your hands).
3. Place on ice, and perform an ice bath for 30 minutes.
4. Perform heat shock at 42°C for 90 seconds.
5. Place back on ice for 2–3 minutes.
6. Add 600–700 μl of LB medium (without antibiotics), and incubate in a 37°C shaker for 50–70 minutes.
7. Centrifuge at 5000 rpm for 5 minutes, discard the supernatant, resuspend the remaining 100 μl by pipetting, and plate the cells on the appropriate antibiotic-resistant agar plate. Incubate overnight at 37°C.

Results:

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The experimental process was lengthy today and could not be completed in time to view the results. The relevant results will be available the next day.

ResultDay 7 – 20240726

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Experiment 6: Bacterial Shaking

Experimental Purpose:

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Monoclonal expansion

Procedure:

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1. Use a sterilized toothpick or Tip pipette to pick a single colony, add it to a single tube containing 4ml of LB medium containing 1‰ of corresponding resistance, place it in a shaker, and incubate it overnight at 37°C at 200rpm to obtain the mother liquor;
2. Aspirate 50~100μl of the mother liquor of the bacteria, add it to a single tube of 4ml of LB medium containing 1‰ of the corresponding resistance and 1‰ inducer (CopyCutter Induction solution), and place it in a shaker for overnight culture at 37°C at 200rpm.

Result

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Day 8 – 20240727

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Protein Expression Assays

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Experimental Purpose:

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To further determine whether PdxA and PdxJ proteins are successfully expressed.

Procedure:

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1. The recombinant plasmid was transferred into Escherichia coli Rosetta (DE3) competent cells, and then plated on plates containing 30µg/mL kanamycin and 34µg/mL chloramphenicol.
2. Monoclonal cells were selected and cultured in liquid medium containing 30µg/mL kanamycin and 34µg/mL chloramphenicol.
3. When the OD value reached 0.6, 0.5mM IPTG was added, and the cells were cultured at 20°C overnight and 37°C for 6h, respectively.
4. The cells were collected by centrifugation, the supernatant was discarded, and the cells were collected.
5. PBS was added to the collected bacteria suspension, which was fully dissolved using an ultrasonic crusher. It was then centrifuged, and the centrifuged precipitate was dissolved using buffer B. The supernatant and precipitate were processed separately to prepare for SDS-PAGE detection.

Results

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Day 9 – 20240728

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GFP fluorescent protein detection

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Experimental Purpose:

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To further determine whether PdxA protein and PdxJ protein are successfully expressed. We inserted the GFP sequence at the end of the PdxA-PdxJ gene, allowing us to observe its expression using fluorescence microscopy. As shown in the figure above, with strong fluorescence intensity, which proved that the expression of the PdxA-PdxJ gene was good.

Experimental Process:

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1. Transformation: The recombinant plasmid was transformed into BL21(DE3) E. coli competent cells. After heat shock at 42°C, the cells were plated on ampicillin-resistant plates (50 µg/mL) and incubated at 37°C.
2. Activation: A single colony was picked and cultured in liquid LB medium with 50 µg/mL ampicillin at 37°C.
3. Induction: When the OD value reached 0.6, 0.5 mM IPTG was added, and the culture was continued overnight at 20°C or for 6 hours at 37°C. The uninduced sample served as the negative control.
4. Collection: Cells were collected by centrifugation (4000 rpm for 10 minutes), and the supernatant was discarded. The cell pellet was collected.
5. Expression Detection: The collected cell pellet was suspended in buffer A and lysed using an ultrasonic crusher. The lysate was centrifuged, and both the supernatant and precipitate proteins were prepared for gel detection.

Main Experimental Results:

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Day 10 – 20240731

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Construction and Verification of Linear Expression Vectors

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Experiment purpose:

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To facilitate the expression and visualization of PDXA and PDXJ proteins, GFP fluorescent protein was used as a reporter gene, producing yellow-green fluorescence. The pRSFDuet-PGFP plasmid was constructed as a positive control.

Experimental process:

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1. This protocol is to add SP6 promoter, E01 translational enhancer, and 3’-UTR sequences by split-primer PCR method to the cDNA which is inserted in a vector other than pEU-E01-MCS vector.
2. If cDNA is inserted in a vector of pET-24 series or pET-28 series, PCR products may not be obtained. The vector containing the cDNA of target protein should not contain SP6 promoter sequence. If it does, it is recommended to change the vector.
3. With PCR DNA, the protein yield is reduced to about 70% of that with plasmid DNA

The PCR procedure follows standard parameters, with one round and two rounds of amplification. The results were checked by agarose gel electrophoresis. Relevant primer sequences are shown in the table below.

1. Using a 0.2-ml PCR tube, prepare a reaction solution of the following composition

   

2. Place the reaction tube in the PCR thermal cycler, program the cycler for the following condition, and run the first PCR campaign.

   

3. Run agarose gel electrophoresis with 2 µl of the PCR product solution to see if the target product is detected as a single band (*4).

(Notes:)

1. Since the volume of required Taq polymerase is small, mix enough amounts of the reagents for at least 10 samples to measure out the correct volume of the polymerase.
2. To adjust the elongation time and temperature according to the DNA polymerase and the length of the gene, follow the supplier’s instruction for the DNA polymerase.
3. Proceed with 2nd PCR even if 1st PCR product is not detected, because 2nd PCR may still yield the product

Main Experimental Results:

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The two genes were first connected, primers were added at both ends, and the sequences were amplified and purified by PCR. A cDNA fragment with a known sequence was obtained, and SP6 promoter and 3’-UTR sequences were introduced using primers. The gel electrophoresis results are shown below.

Day 11 – 20240803

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Cell-Free Expression System

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The validated linear expression vector was added to the reaction system. The expression of target genes was evaluated by monitoring the green fluorescent protein. As shown below, after some time, the supernatant turned yellow-green, indicating the expression of the target protein.

Experimental Process:

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Step 1: Concentration of PCR Product

1. Transfer the whole content of 2nd PCR product solution into a 1.5-ml tube.
2. To this 2nd PCR product solution, add 100% ethanol, 2.5 times the volume of the solution, and 3 M Sodium acetate, one tenth of the volume (*1).
3. Mix the solution well and incubate for 10 minutes at -20 °C.
4. Centrifuge the mixture at 15,000 rpm for 15 minutes at 4 ˚C.
5. Remove the supernatant and add 300 µl of 70% ethanol to it.
6. Centrifuge the mixture at 15,000 rpm for 5 minutes at 4 ˚C.
7. Remove the supernatant as much as possible and dry it for 15 minutes (2).
8. *Add 1/10 volume (of the 2nd PCR product solution when transferred into a 1.5-ml tube in the first step) of nuclease-free water. Let the tube stand for 10 minutes to loosen the DNA pellet, and then resuspend the pellet gently by pipetting (*3).
9. Run agarose gel electrophoresis with 0.1 µl of the solution to confirm the presence of the concentrated PCR product. Two microliter of the concentrated PCR product is required for the transcription reaction.

*1. Do not dilute the 2nd PCR product solution before adding ethanol and sodium acetate.

*2. The DNA pellet should not be too dry to resuspend.

*3. It may be possible to increase the protein synthesis yield by increasing the concentration of PCR product

Step 2: Transcription of PCR DNA into mRNA

1. Remove from storage (-80 °C) required number of Transcription Premix LM tubes (blue) (*1). Keep the remaining tubes in storage at -80 °C.
2. Thaw the Transcription Premix LM on ice. After thawing, spin down the tube for a short time to drop down the reagent staying on the tube wall or on the cap.
3. Add 2 µl of the concentrated PCR product to each tube of the Transcription Premix LM and then mix gently by pipetting.
4. Incubate at 37 °C for 4 hours in a thermal cycler or an incubator (*2).
5. After the incubation, inspect mRNA quality by the ordinary method of agarose gel electrophoresis with 1 μl of the mRNA sample (*3)

Step 3: Translation of Target Protein

1. Remove from storage (-80 °C) required number of WEPRO®9240/9240H/9240G tubes and single-break strip wells (clear) containing SUB-AMIX® SGC (*1). Keep the remaining tubes and wells in storage at -80 °C
2. Thaw the two reagents on ice. After thawing, spin down the tube containing WEPRO®9240/9240H/9240G for a short time to drop down the reagent staying on the tube wall or on the cap. Avoid excessive centrifugation. Resuspend SUB-AMIX® SGC by pipetting gently in the well (*2).
3. Let the mRNA tube(s) cool down to the room temperature. DO NOT forcibly cool it on ice or in the refrigerator. Resuspend the mRNA by pipetting gently (*3).
4. Add 10 µl of resuspended mRNA into WEPRO®9240 and then mix gently by
5. pipetting. Avoid bubble formation.
6. Carry out bilayer reaction.
7. Carefully transfer the whole mixture (20 μl) of WEPRO®9240/9240H/9240G and mRNA to the bottom of the single-break strip well containing SUB-AMIX® SGC (206 μl) to form bilayer with WEPRO® mixture in the lower layer and SUB-AMIX® SGC in the upper layer as illustrated below. DO NOT mix the reagents in the well by pipetting or any other means. (Important !!)
8. Seal the well with aluminum seal included in the kit to avoid evaporation (*4).
9. Incubate at 15 °C for 24 hours.
10. After translation, mix the bilayer reaction gently by pipetting.

Main Experimental Results:

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Day 12 – 20240804

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Experiment purpose

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To optimize the yield of PDXA and PDXJ protease in a cell-free expression system by adjusting reaction times and temperatures. The reactions were performed at 37°C, and the fluorescence absorbance was measured at different time intervals using a spectrophotometer.

Experimental Procedure:

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Adjust the reaction tube’s temperature. Set reaction times at 0.5, 1, 2, and 3 hours. Collect samples at each time point, and measure the fluorescence absorbance using a spectrophotometer. Each measurement was repeated three times, and the trends were plotted, analyzing the statistical significance. The spectrophotometer was operated according to the standard manual.

Experiment results:

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Day 13 – 20240805

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Experiment purpose:

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Based on the previous time optimization, further optimize the reaction temperature for maximum expression of PDXA and PDXJ proteins.

Experimental Procedure:

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Adjust the reaction tube to temperatures of 15°C, 25°C, and 37°C. Conduct the expression reaction at these temperatures, and use a spectrophotometer to measure absorbance. Each measurement was repeated three times, and the results were analyzed for trends and statistical significance. Spectrophotometer use followed standard methods.

Experiment results:

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Day 14 - 20240806-0807

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To assess the effectiveness of VB6 biosynthesis and compare the results of the cell-free system to those of E. coli strains.

Experimental Procedure:

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Following the previous experimental steps, samples were collected at 1, 2, 4, 8, and 12 hours. The substance concentration in each sample was measured using a spectrophotometer, and the VB6 relative concentration was calculated for comparison. The spectrophotometer was used according to the standard test manual.

Results:

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The results showed that the reaction reached its peak at 6 hours in vitro, producing up to 30 mg/L under small-scale expression conditions. Compared to intracellular E. coli expression systems, the yield was significantly increased (p=0.0109, as shown in Figure 13). This suggests the potential for a faster and more efficient green production system for VB6.

Day 16 – 20240810

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Experiment purpose:

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To further verify the feasibility of the synthesis system, HPLC was used to analyze the reaction products.

Experimental Procedure:

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Step 1: Preparation–Instrument Setup

1. Turn on the HPLC, including the pump, detector, and autosampler (if applicable), and preheat the system to a stable working temperature (e.g., a column oven temperature of 40°C).
2. Ensure there is sufficient mobile phase in the storage container. Depending on experimental requirements, prepare different proportions of mobile phases such as A (acetonitrile) and B (aqueous solution with additives like 0.2% phosphoric acid and 5 mM IPCC-06).
3. Check all connections for leaks, and install the selected column (e.g., Inertsil ODS-3, 5 μm, 150 × 4.6 mm I.D.) in the column oven according to the manual.
4. Balance the column using a low flow rate (0.5–1.0 mL/min) with the initial mobile phase ratio (e.g., A/B = 5/95) until the baseline stabilizes. This may take 30 minutes to several hours, depending on the column type and previous usage.

Step 2: Sample Preparation

1. Prepare the sample solutions for analysis. Ensure the samples are fully dissolved and free of suspended particles, using filtration or centrifugation if necessary. Transfer the samples to appropriate vials and label them clearly. For autosampler use, load the vials into the designated tray and set the sample location and injection volume (e.g., 20 μL) in the instrument software.

Step 3: Experimental Operation

1. Set instrument parameters in the HPLC software, including flow rate (e.g., 1.0 mL/min), detection wavelength (e.g., UV 210 nm), column temperature (e.g., 40°C), and the gradient program for the mobile phase (e.g., A/B = 5/95 initially, gradually changing to 20/80 over a certain time).
2. Set appropriate run time and data acquisition frequency.
3. Start the autosampler or manually inject the sample into the column.
4. Observe the chromatogram in real-time, recording retention time, peak area, peak height, and separation resolution.

Step 4: Post-Analysis System Cleaning

1. After analysis, flush the column and system lines with a high proportion of the organic phase (e.g., 100% acetonitrile) to remove residual samples and impurities. Adjust the flush time and flow rate based on the situation.
2. Rebalance the column with the initial mobile phase ratio for the next analysis.
3. Turn off the system components (pump, detector, autosampler) in sequence.
4. Depending on the properties of the remaining mobile phase, decide whether to replace or store it for future use. Clean the workstation and organize the supplies.

Results:

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