Team:LZU-HS-CHINA/Notebook

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NOTEBOOK OF LZU-HS-CHINA

NOTEBOOK of Reduction of selenite in vitro by EcN-IS cell biocatalyst

Project: LZU-HS-CHINA IGEM 2021

Dates: 2021-05-03 to 2021-07-25

The First Week

6.14-6.20

Laboratorian: Colin Liu, Maggie Shen
Others: Team

Preparation

  • Prepare for the material required in next week.
  • Make sure all the reagents are available in the laboratory.
  • Check all the devices to ensure they are in a good condition.
  • Study the safety requirements and have lab skills training.

    • Research

  • Write the experimental plan for the whole season, examined by instructor Liu Jinrong.
  • Read thesis and papers about the reduction of Selenite and the synthesis of SeNPS.
  • Read thesis about the use of Nissle 1917.
  • Read thesis about Microbial Cell Surface Display System.

    • Human Practice

  • Design Investigation questionnaire
  • Make appointment with Ms.Guo and Ms.Chen

    • Collaboration

  • Make appointment with ShanghaiTech-China, SJTU-BioX-China
  • Meet XHD-Wuhan-Pro-China, Think-Edu-China

    • The Second Week

    6.21-6.27
    Laboratorian: Matthew Zhang, Maggie Shen
    Preparation and Activation

    1. Sterilization: Put all needed items(medium and experimental supplies) in at 121℃ for 20 min.
    2. Dilution primer: Primer dry powder was centrifuged, and water labeled on the tube wall was added to obtain mother liquor (100 mM). Mother liquor was diluted 10 times with ddH2O.
    3. Amp antibiotic mother solution (100 mg/mL): Accurately weigh 1g Amp powder in a glass, add 10 mL dH2O to dissolve it, filter it into a sterile 2 mL EP tube with a filter, and store it at minus 20℃.
    4. Activate 4 tubes of DH5α/ PSB1A3-MRFP and 2 tubes of EcN: 100µl of stored bacteria solution was added to each test tube (2.5µl Amp antibiotic was added) The plugged test tube was bound and placed in a shaker for overnight culture (150 RPM, 37℃).
    5. Ser was amplified with high fidelity enzyme, EcoRI and SpeI were introduced into the enzyme digestion site, 4x50 µL reaction. PCR products were stored in refrigerator at 4℃

    The Third Week

    6.28-7.4
    Laboratorian: Tengge Wang, Yikai Yang
    Extraction and Activation

    1. Prepare 50 mL 1% agarose gel
    2. Extract 4 tubes of DH5α/ Psb1a3 MRFP plasmid according to the instructions of Plasmid Extraction Kit. The recommended elution concentration is 50 µL. The extracted plasmid psb1a3 MRFP and the inserted gene ser were double digested by EcoRI and spei. 5x20 µL reaction. Enzyme-cutting for 4 hours
    3. Extract ECN genomic DNA according to the instructions of DNA extraction kit.
    4. The ECN 16S gene (2H) was amplified with KOD enzyme, 2 x 50 µL reaction system
    5. Add PSB1A3-mRFP and ser enzyme products and EcN 16S amplification products into agarose gel.
    6. Add 5µL DL 2000 and DL 5000 DNA markers.
    7. Perform electrophoresis (110V, 45 min). After the end of electrophoresis, the gel system was used to observe the target. Cut DNA gel and place it in a 2 mL centrifuge tube for marking. 8. Recover the gel of DNA according to the instruction of adhesive Recovery Kit (1H). Preserve DNA with - 20 ℃.
    8. Activate each tube of dh5a and ECN (150 rpm, 37 ℃).

    The Fourth Week

    7.5-7.11
    Laboratorian: Tengge Wang, Colin Liu
    Ligation and Transformation

    1. Prepare for required material to ensure they are in the condition that is proper for experiment
    2. Link The double enzyme digestion product psb1a3 with Ser. Connect the amplified product 16S with the linearized pmd-18t (commercial) vector (15min). Use PCR instrument to connect at 16 ℃ for 1H. The linked products were stored at - 20 ℃.
    3. Prepare LB solid medium (add 4.5 g agar to 300 ml LB liquid medium), and sterilize at 121 ℃ under high temperature and high pressure for 15 min. After cooling to about 60 ℃, add 150 µL 100mg / ml amp antibiotic, shake evenly and pour it into a disposable Petri dish (about 10 plates can be poured)
    4. Prepare the receptive state of dh5a, 1917 (3H, 2h free in the middle).
    5. Add 10 μL psb1a3-ser and pmd18t-16s to 100 μL receptor cells for 30min in an ice bath, 60s in a 42℃ water bath, 5min in an ice bath, and 1 mL LB liquid medium for 1h for recovery (37℃, 150 RPM). In the ultra clean table, take 100 μL of recovery solution and use sterile coating rod to coat Amp antibiotic plate.

    The Fifth Week

    7.12-7.18
    Laboratorian: Hedia Wei, Matthew Zhang
    Cloning

    1. Add 2.5µl amp antibiotics to LB tube, pick three tubes of dh5a / pmd18t-16s colonies into LB Test tubes and culture overnight in a shaking table (37 ℃, 150 RPM).
    2. Mark 6 DH5 α/ Psb1a3 ser colony. Half of the colonies were selected as the template and Taq enzyme was used for colony PCR. The primers were VF and VR, and the reaction system and procedure were as described above. 50 mL agarose gel was prepared and electrophoresis was performed using DL 2000 DNA marker.
    3. Pick and verify the correct DH5α/ Psb1a3 ser colony was added to LB liquid medium and 2.5µl amp antibiotics, expanded culture.

    The Sixth Week

    7.19-7.25
    Laboratorian: Colin Liu, Matthew Zhang
    Transformation and Checking

    1. Add pSB1A3- ser to EcN
    2. Grow ECN-IS, ECNPSB1A3 in LB plates to OD600=0.6
    3. Plant 1%(Vbacteria/VLB plate) on 100ml LB plates with 1mM Na2SeO3
    4. Grow in 180rpm for 24h
    5. Take out 5ml bateria in each 4h from each plate.
    6. Centrifuge the supernatants at 12000rpm to measure the amount of residue of Na2SeO3 through ICP-OES
    7. Check the bacteria growth concentration in plates for each 4 hours by measuring the change of OD600 value