Date: 8.3
      Date: 8.4
      Date: 8.5
      Date: 8.6
      Date: 8.7
      Date: 8.8
      Date: 8.9
      Date: 8.10
      Date: 8.11
      Date: 8.12
      Date: 8.13-8.23
Date: 8.3
Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li, XueSong Lu, Yuncong Dong, YuFei Li
Procedures
  • Rinsing Equipment
    inse all equipment with double-distilled water.
  • LB broth Preparation
    Dissolve 25 g of LB powder in 1 liter of water, adjust pH to 7.0, and autoclave at 121°C for 15 minutes.
  • Solid Medium Preparation
    Add 15 g of agar to the LB broth, pour into petri dishes, and allow to solidify.
  • Sealing
    Seal conical flasks with parafilm and rubber bands.
  • Sterilization
    Autoclave sealed flasks at 121°C for 20 minutes.
Date: 8.4
Participants: Zhichen Jin, Wenbo Xu, Yixin Yan, Shuhan Hu, Yi Zhang, Yuxin Wu
Procedures
  • ET Bacterial Culture
    Inoculate E. coli into shaking culture tubes containing LB liquid medium with ampicillin resistance under a clean bench. Incubate overnight in a 37°C shaking incubator. The following day, the LB broth should appear turbid, indicating successful amplification of E. coli.
  • Gel Recovery
  • Plasmid Extraction
  • Configure PCR System
  • Configuration of Agarose Gel
    Weigh 0.5 g of agarose powder and add it to a 50 mL conical flask containing 50 mL of TAE buffer.
  • Run the Gel Turn on the electrophoresis tank, set it to 180 V, and run for 30 minutes. Then, turn off the electrophoresis tank.
  • Gel Imaging
    Transfer the gel to the gel imager for UV irradiation and observe the band sizes. The results are as follows:
  • Cutting Gel
    Excise the agarose containing the target gene under external light.
Date: 8.5
Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li
Procedures
  • Measuring pRSF Sample Using Nanodrop
    Fig.1 At a wavelength of 260 nm, the absorbance of pRSF is 0.349; at 280 nm, it is 0.197; and at 230 nm, it is 0.322. The concentration of DNA in pRSF is 17.45 ng/µL.
  • Cutting the pRSF Plasmid
  • Digestion of the metF Gene and ftfL Gene
  • Gel Electrophoresis
  • Analysis of Gel Electrophoresis
    In addition to conventional annotation, the brightness of the electrophoretic image should be increased without any retouching.
    Fig 2. The first result is the marker; the second is pRSF, and the third and fourth results are for the metF gene.
  • Preparation for Recycling of Agarose
    Ensure that the washing buffer contains 45 mL of absolute ethanol.
  • Recycling of Agarose
  • Combining pRSF and metF gene, pET and ftfL Gene
Date: 8.6
Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li
Procedures
  • Recombinant Plasmid Transformation in the E. coli DH5α
    Put the solution on a petri dish with a solid LB broth with kanamycin Put in a 37°C incubator
Date: 8.7
Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li, XueSong Lu, Yuncong Dong, YuFei Li
Procedures
  • Preparation of Mixture for 5 Single Colonies
    Add 90 μL of 2× Mix Star, 72 μL of ddH₂O, and 6 μL each of Met-F and Met-R to a small tube. Gently rotate the tube to mix. For the second mixture, combine 90 μL of 2× Mix Star, 72 μL of ddH₂O, and 6 μL each of FtfL-F and FtfL-R in a separate small tube. Gently rotate the tube to mix.
  • Extraction of Single Colonies
    Transfer the colonies to the prepared mixtures.
  • Polymerase Chain Reaction (PCR)
    Prepare the gel for electrophoresis.
  • Gel Electrophoresis
    Perform gel electrophoresis to verify whether the target fragments have been successfully transferred into E. coli.
Date: 8.8
Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li, XueSong Lu, Yuncong Dong, YuFei Li
Procedures
  • Plasmid Extraction and Enzyme Digestion Verification
    Combine 5 µL of pRSF-metF-1, pRSF-metF-2, and pRSF-metF-3 with 3 µL of 10× green buffer, 1 µL of BamHI, 1 µL of HindIII, and 20 µL of ddH₂O in separate PCR tubes (30 µL per tube) using pipettes.
  • Selection of the Correct Plasmid and Target Gene folA for Double Enzyme Digestion Ligate folA to pRSF-metF and culture overnight at 16°C. Similarly, ligate mtdA-fchA to pET-ftfL and culture overnight at 16°C.
  • Enzymatic Digestion and Gel Recovery
Date: 8.9
Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li, XueSong Lu, Yuncong Dong, YuFei Li
Procedures
  • Transformation in E. coli BL21 (DE3)
  • Preparation of the LB broth
Date: 8.10
Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li, XueSong Lu, Yuncong Dong, YuFei Li
Procedures
  • Preparation of Mixtures for Single Colonies
    Add 90 μL of 2x Mix Star, 72 μL of ddH₂O, and 6 μL each of folA-F and folA-R to a small tube. Gently rotate the tube to mix. Prepare another mixture by adding 90 μL of 2x Mix Star, 72 μL of ddH₂O, and 6 μL each of mtdA-fchA-F and mtdA-fchA-R to a separate small tube.
  • Extraction and Transfer of Single Colonies
    Extract single colonies and transfer them to the prepared mixtures.
  • PCR
    Perform PCR as needed.
  • Preparation of Gel for Electrophoresis
    Prepare the gel for electrophoresis.
  • Gel Electrophoresis
    Conduct gel electrophoresis to verify the presence of target fragments transferred into E. coli.
  • ET Bacterial Culture
    Inoculate E. coli into shaking culture tubes containing LB liquid medium with ampicillin resistance under a clean bench. Incubate overnight in a 37°C shaking incubator. The following day, the LB broth should be turbid, indicating successful amplification of E. coli.
Date: 8.11
Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li, XueSong Lu, Yuncong Dong, YuFei Li
Procedures
  • Preparation of SDS-PAGE
  • One-step Growth Curve Measurement
    Sampling and measuring the bacterial culture's OD600 every two hours for 12 consecutive hours.
  • A 250 mL culture of LB broth, supplemented with the appropriate antibiotics, was inoculated with the bacterial culture and incubated on a shaker at 37°C until the OD600 reached a value between 0.5 and 0.6. Subsequently, IPTG was added to the medium, and the culture was further incubated overnight on a shaker at 18°C.
Date: 8.12
Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li, XueSong Lu, Yuncong Dong, YuFei Li
Procedures
  • Bacterial cultures were harvested by centrifugation.
  • Ultrasonic cell disruption was performed for protein extraction.
  • The protein samples were loaded onto an SDS-PAGE gel for separation, followed by staining with Coomassie Brilliant Blue and subsequent destaining.
Date: 8.13-8.23
Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li, XueSong Lu, Yuncong Dong, YuFei Li
Procedures
  • Compiling and analyzing experimental data into a coherent format, followed by the composition of a comprehensive wiki entry detailing the findings and methodology