Team:Saint Joseph/Experiments

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Experiments

LB Broth Media Preparation

Growth Medium Preparations Luria-Bertani (LB) medium is used for the establishment of cell cultures in all experiments to be carried out.

  • Preparation of LB Broth (Liquid) Media
    1. 25 grams of LB Broth Powder (Tripton, Yeast Extract, NaCl 2:1:2) poured into a bottle with a sterile screw capped bottle.
    2. Complete the volume with 1 liter of ddH2O. Cover it halfway and wrap it with foil autoclave tape.
    3. Autoclave at 121°C for 15 minutes.
    4. After cooling at room temperature, store at 4°C.
  • Preparation of LB Agar (Solid) Medium
    1. Prepare 1 Liter of LB broth in a 2-liter flask using the first two steps described above. Add 15 grams of bacterial agar (1.5%).
    2. Autoclave at 121 °C for 15 minutes.
    3. Cool down to approximately 50 °C.
    4. If antibiotics are to be added, they must be added at this stage with the right temperature, otherwise the antibiotic added to a warm environment will degrade rapidly. Mix by vigorously rotating the bottle.
    5. The medium is poured into petri dishes. When pouring the petri dish, be careful not to drop the temperature too much.
    6. Approximately 25-30 ml of LBA can be poured into a petri dish.

We started the transformation experiments by preparing competent cells according to the protocol. We used E. coli DH5a bacteria. We inserted plasmid DNA to these bacteria by heat-shock transformation.

Competent Cell Recovery and Transformation

  • We ordered our plasmids from IDT
  • Making E. coli DH5α strain competent (with CaCl2 Method)
    A single colony is taken from the E. coli DH5α cultured solid medium and inoculated into 10 ml of LB medium. The culture is incubated at 37°C overnight. Then, overnight culture is incubated on ice for 10 minutes. It is centrifuged at 14000 rpm at 4°C for 5 minutes in a refrigerated centrifuge and the supernatant is discarded. 2 ml of 0.1 M CaCl2 added to the pellet and keeped on ice for 30 min. It is centrifuged at 14.000 rpm at 4°C for 5 minutes in a refrigerated centrifuge and the supernatant is discarded. 0.4 ml of 0.1 M CaCl2 added to the pellet and stored at 4oC (it must not be kept no longer than 18 hours.) Each transformations made by 200 microliters of these competent cells bu using heat shock.
  • Plasmid isolation (For stockpiling, with strain DH5α)
    The plasmid isolation is done with the Thermofisher GeneJet Plasmid Miniprep kit, according to the protocol recommended by the manufacturer.

    Pellet bacteria by centrifugation, Add Lysis Buffer followed by Neutralizing Buffer, Centrifuge to pelletet protein precipitate, Transfer supernatant to GET Spin Column and centrifuge, Wash and elute plasmid DNA
  • Glycerol stock purchases
    Prepared with 50% glycerin distilled water. The overnight grown cell culture is mixed 1:1 with 50% glycerol. Store at -80 °C.

Next, we needed to insert the desired piece of DNA into our plasmids by enzyme cutting.

  • Recovery from Gel (with GF-1 DNA Recovery kit)
    Digested DNA fragments separated and isolated from agarose gel with GF-1 DNA Recovery Kit according to the manufacturer’s instructions After cutting, we linked the additional DNA to our vectors by ligation.
Ligation

Vector and Insert Preparation and Ligation

Depending on whether the insert DNA sequence is obtained as a DNA fragment or in the Cloning vector, it is necessary to transfer this sequence to an Expression vector for the protein expression. With this aim, the gene is cutted from the restriction enzyme sites (different from each other) placed at the beginning and end of the sequence while designing the sequence, using appropriate enzymes. The expression vector to which the sequence is to be transferred is also cut from the same enzyme cut sites. Then, the insert gene ligated with the pET29b expression vector and adhered.

Preparation of plasmids

Once the desired result was achieved, it was time to screen the plasmids from the gel. We followed the protocol in these parts.

Insert DNA plasmid backbones that isolated from the agarose gel were ligated with T4 DNA polymerase and transformed into E. coli cells.

We ran our plasmids in agarose gel electrophoresis to see if the ligation was successful.

Agarose Gel Electrophoresis

After ligation and transformation, transformant cells obtained at the LBA+Kan selective media. For selecting the recombinant cells, transformant colonies are analyzed by colony PCR with T7 forward and reverse primers, according to the tables below. After the colony PCR experiment, samples were analyzed by agarose gel electrophoresis. And the obtained recombinant colonies inoculated into LBB+Kan media overnight. After the incubation, plasmids isolated from these cultures. Isolated plasmids were analyzed with cutting with restriction enzymes at the agarose gel.

Then, we again produced competent cells. Thus, we introduced our expression-ready vector into DH5a and BL21 cultures by transformation so that it could grow.

  • Production of competent cell and transformation
    In order to multiply the vector that is ready for expression, it must be administered in the E. coli DH5α strain. For the expression, the vector must be given to E. coli BL21(DE3) strain. Induction of protein expression and isolation For the targeted gene in the vector that has been administered to the expression strain to be expressed, protein expression must be triggered by the inducers that will join the environment. In these experiment protocols, usage of pET expression vectors that ensures high expression has been planned. In these vectors that the gene expression is executed under T7 promoter controle, IPTG or lactose can be administered in the medium as a trigger of gene expression.
    Underlying mechanism of induction can be explained: The lac repressor, constantly being produced by the lacI gene that is located in E. coli genome, inhibits the expression of T7 gene that executes the T7 Polymerase production by preventing the necessary polymerase in gene expression to stick to the gene. By the addition of IPTG in the medium, the IPTG attaches to the produced lac repressor and prevents the inhibition of T7 polymerase production; therefore, T7 polymerase is produced and gene expression under T7 promoter controle can be executed.

    IPTG process

Induction of protein expression

  • Production of competent cell and transformation
    A colony is selected from the fresh petri and amplified under 30°C 2mL in LBB+Kan overnight. Overnight culture is diluted with LB+Kan by 1:50 ratio as it will be 5mL.
    It is produced in 37°C for 3 to 4 hours. Meanwhile, the medium that contains 1mL LB + antibiotic + 1mM IPGT is heated to 37°C. 1mL culture and 1mL LB + Kan + IPGT is mixed. It is incubated for 3-4 hours. Cells are separated by 1mL and collected by centrifuge. Pellet is kept until used at -20.

    As protein synthesis can be extremely fast in temperatures higher than 25-30°C, ratio of misfolded protein can increase. Optimisation is needed.

Protein Induction

  • Protein Isolation
    Protein isolation can be performed by the help of 6x Histidine Tag that is added to the 3’ end during the genetic design process. It is made by using Promega MagneHisTM Protein Purification System kit.

Protein Isolation by magnets

Bradford Analysis

We used Bradford analysis which is used to determine the protein concentration.
In order to create Lyophilized BSA standard, 20mL deionised water is added and stirred until dissolved/ If the standards will not be used within 60 days, they are aliquoted and stored in -20°C.

Note: Standards contain the necessary buffer salts for the solubility of protein

For the quantification of pure enzyme samples isolated from E. coli BL21 bacteria, analysis was performed using the Bradford method. During sample preparation for Bradford analysis, “Bio-Rad Protein Assay Kit II” colorimetric protein measurement kit (Bio-Rad, 5000002) and BSA (Bovine Serum Albumin) was used as a standard. A stock solution of 1 mg/mL BSA was obtained by adding 20 mL of deionized water to lyophilized BSA. Five BSA standards were prepared at 0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL concentrations by diluting the final volume to 100 microliters. To prepare the dye reagent solution "Dye Reagent Concentrate" in the kit for spectrophotometric measurement, 400 microliters of dye reagent was diluted with 1.6 mL of distilled H2O. 900 microliters of deionized distilled H2O was added to each of the purified protein solutions containing the purified protein and having a final volume of 100 microliters and these samples were used in the analysis. 10 microliters of each of the BSA standard solutions and sample solutions were pipetted and loaded into the wells on the 96-well microplate. Then, 200 microliters of diluted dye reagents were added to each well and pipetted until all solutions became homogeneous. Each sample was loaded into the wells in duplicate. The microplate was incubated for 10 minutes at room temperature. The microplate was placed in the "Thermo Scientific Multiscan GO Microplate Spectrophotometer" device and absorbance was measured at 595 nm. The BSA standard graph was created with the data obtained after the measurement. The protein concentration of the samples was calculated by taking the BSA standard

Enzyme activity control

pNP-cellobiose or carboxymethylcellulose can be used as substrates.

Enzymatic activity is predicted with the DNS method. The DNS method can be actualised with tracing the reduction of sugars like carboxymethylcellulose (CMC). Reduction of glucose or other oligosaccharides turns the DNS (yellow) to 3-amino-5-nitrosalicylic acid (orange-red). Change in colour can be measured as absorbance with a spectrophotometer; thus, it can be used to determine the activity of endoglucanase that produces the reducing sugars.