Team:MSP-Maastricht/Experiments

Experiments

Brainstorming Stage

An article by Thapa et al. (2020), which found the genetic mechanism for bromoform production in various seaweed species inspired us. The suggestion that feeding cows seaweed, such as A. taxiformis would reduce the amount of methane (CH4) produced from cows (Roque et al., 2019) initiated our research and led us to Thapa et al. (2020). This was fascinating and we investigated further. The algae A. taxiformis contains specialized gland cells from which the bromoform is secreted as a defense mechanism against parasites and herbivores (Machando et al., 2016). The bromoform can be produced through a photosynthetic-derived pathway or via a fatty acid biosynthesis-derived pathway (Zhu et al.,2021). Thapa et al. (2020) and Thapa & Agarwal (2021) explored the usage of pentane-2,4-dione and heptane-2,4,6-trione as substrates for the synthesis of bromoform. The genes in question were mbb1 and mbb4 which had the highest levels of bromoform production. The mbb1 and mbb4 are vanadium-dependant haloperoxidases (VHPOs) and they require a reactive oxygen species as a cofactor to generate the bromoform. Thus the solution for the creation of the ROS was also present as a gene in A. taxiformis – mbb2. Furthermore, VHPOs were present in another algae species, namely Chondrus crispus (Thapa et al. 2020). Two bromoform producing genes were identified – CcVHPO1 and CcVHPO3 which were highly efficient. Therefore, once we had conceptualized and actualized our idea with the genes, we wanted to test forward with the designing process.

Schematic of bromoform synthesis pathway
Hypothesized Pathway of Bromoform Biosynthesis with Substrates and Products in Green and Enzymes shown in Yellow. Adapted from Zhu et al. (2021) and Thapa et al. (2020).

Design Stage

The DNA sequence of the genes was retrieved from Genbank. From this we wanted to maximize the number of combinations with the minimal number of genes required to be processed. We wanted a device that would allow us to orient the genes in whichever pattern we wanted. To do so we create the Spacer concept, essentially a simple DNA sequence that does not code for any proteins, nonsense DNA, however that would contain valuable recognition sequences. It contained a ribosome binding site to maximize the efficiency of large, transcribed proteins. Along with the RBS there was a restriction site XbaI introduced for both gel electrophoresis detection and analysis as well as being able to insert the gene of interest after the spacer for usage if so required. Then we wanted another spacer to flank the end of the target gene of interest. The back spacer contained nonsense DNA that would not be able to produce a protein. Again, for testing purposes as well as inserting the genes, a restriction site was present, NdeI.


Experimental Stage

For this we used several protocols. We followed this process:

  • Creation of the competent cells – DH5a for cloning and BL21 for protein expression
  • Assembly of the single genes in the DH5a cells. To maximize the usage of DNA that was generated we inserted the gene of interest into DH5a so that with a correct transformation using Gibson Assembly we would be able to generate plates containing that construct which we can use whenever and in an endless supply
  • After every Gibson Assembly we always made sure that we would run an agarose gel to be able to analyze what we were able to construct.
  • Following this we created our pattern system using the spacer technology to create our gene orientations
  • Protein Secretion
  • Protein Purification
  • Testing in cow ruminal fluid (see Model section)


Chemically Competent Cells



Materials
  • Overnight Culture of cells of your choice
  • Ice-Cold 0.1M CaCl2
  • Ice-Cold 0.1M CaCl2 +15% glycerol
  • 50ml Falcon tubes
  • Pre-chilled Eppendorf tubes
Procedure
  1. Create an overnight culture in LB liquid without any antibiotics
  2. Grow the cells in a incubating shaker (250 to 270rpm) at 37C until the OD600 of the cells is between 0.4-0.6
  3. Chill the cells on ice for 10 to 15 mins (maximum is 1 hour do not exceed this time) to prevent further growth
  4. Spin down the cells in a 50ml Falcon tube at 4000 rpm for 10min at 4C
  5. Remove the supernatant; from now work is to be carried on ice
  6. Suspend the pellet with cold 10ml 0.1M CaCl2
  7. Spin it again at 4000rpm at 4C for 10 min
  8. Gently resuspend cells with cold 2ml 0.1M CaCl2 and 2ml of 15% glycerol
  9. Allocate the solution (200ul) into different pre chilled Eppendorf tubes


DPNI Treatment


Materials

  • CR reactions
  • DPNI vial

Procedure

  1. Add 1µL of DpnI to finished 50µL PCR reactions (or .5µL to 25µL reactions). Pipet or invert to mix.
  2. Incubate the mixture at 37°C for 1-2 hrs (depending on your paranoia or need to remove template DNA).
  3. Alternatively, the solution can be left overnight at room temperature. Periodic mixing may aid digestion (but is unnecessary).
  4. PCR cleanup or gel-purify the reaction for downstream processes. It's that easy!


LB Medium


Materials

  • Tryptone
  • Yeast Extract
  • NaCl
  • Agar

Procedure

  1. LB Agar Plates
  2. Add the following to a 1000ml container:
  3. 10 g tryptone (Fisher, cat. no. DF0123-17-3)
  4. 5 g yeast extract (Fisher, cat. no. DF0127-17-9)
  5. 5 g NaCl (Fisher, cat. no. S271-500)
  6. 15 g agar (Fisher, cat. no. DF0140-07-4)
  7. Adjust volume to 1000 ml with double-distilled water
  8. Adjust to pH 7.0
  9. Autoclave, let cool to about 60°C (cool enough to handle without gloves), and then pour into sterile plastic petri dishes (Fisherbrand, cat. no. 08-757-12)
  10. Allow the plates to cool and solidify at room temperature
  11. Store the plates inverted in a plastic sleeve up to 6 months at 4°C

LB Broth

  1. Add the following to a 1000ml container:
  2. 10 g tryptone (Fisher, cat. no. DF0123-17-3)
  3. 5 g yeast extract (Fisher, cat. no. DF0127-17-9)
  4. 5 g NaCl (Fisher, cat. no. S271-500)
  5. Adjust volume to 1000 ml with double-distilled water Autoclave
  6. Store indefinitely at room temperature


Gel Extraction Kit


Procedure - Extracted from QIAquick Gel Extraction Kit

  1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
  2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg gel ~100 μl). The maximum amount of gel per spin column is 400 mg. For >2% agarose gels, add 6 volumes Buffer QG.
  3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (like Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.
  4. Add 1 gel volume isopropanol to the sample and mix.
  5. Place a QIAquick spin column in a provided 2 ml collection tube or into a vacuum manifold. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum to the manifold until all the samples have passed through the column. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes >800 μl, load and spin/apply vacuum again.
  6. If DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 500 μl Buffer QG to the QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.
  7. To wash, add 750 μl Buffer PE to QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube. Note: If the DNA will be used for salt-sensitive applications (e.g., sequencing, bluntended ligation), let the column stand 2–5 min after addition of Buffer PE. Centrifuge the QIAquick column in the provided 2 ml collection tube for 1 min to remove residual wash buffer.
  8. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
  9. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 μl Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.
  10. If purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.


General Transformation


Materials

  • Competent E.coli Cells - homemade or commercial
  • DNA for insertion

Procedure

  • Thaw a tube of Competent E.coli cells on ice for 10 mins
  • Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
  • Place the mixture on ice for 30 minutes. Do not mix.
  • Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
  • Place on ice for 5 minutes. Do not mix.
  • Pipette 950 µl of room temperature SOC or (LB could work in this case) into the mixture.
  • Place at 37°C for 60 minutes in the shaker-incubator.
  • Warm selection plates to 37°C - the ones that contain Kanamycin
  • Spread 50-100 µl from the Eppendorf container onto a selection plate (make multiple) and incubate overnight at 37°C.



    • Gibson Assembly


    Materials

      NEBuilder HiFi DNA Assembly DNA fragments

    Procedure

    1. Mix 10ng – 100ng of each of the DNA fragments together into 5ul total volume. The length of each fragment and the concentration of the fragments must be taken into account
    2. If using the 2x Gibson Mater Mix from NEB – add 10ul of total DNA to a 10ul Gibson Assembly Master Mix
    3. Mix well by pipetting
    4. Incubate the reaction at 50C for 1 hour



    Glycerol Stock


    Materials

    • 50% glycerol solution
    • Desired cell culture to create stock

    Procedure

    1. Overnight liquid culture creation
    2. After you have bacterial growth, add 500ul of the overnight culture to 500ul of 50% glycerol in a 2ml screw top tube
    3. Dilute pure glycerol in distilled water to create a 50% glycerol solution - so 50:50 ( glycerol:water)
    4. Freeze the glycerol stock at -80C



    Gel Electrophoresis


    Materials

    • Standard 1% Agarose Gel
    • Agarose: 1g
    • TAE: 100ml
    • GelRed® Nucleic Acid Gel Stain: 2-3ul

    Procedure

    Pouring a Standard 1% Agarose Gel:
      Measure 1g of agarose
    1. Mix agarose powder with 100ml 1xTAE in a conical flask
    2. Microwave for 1-3 min until the agarose is completely dissolved – don’t let it overboil however make sure
    3. that there are no white particles in it; caution the contain is hot use thermal gloves
    4. Let the agarose solution cool down to 50C – occasionally test it with the back of your hand for 5 secs
    5. Add 2-3ul GelRed Stain to the agarose solution
    6. Pour the agarose into a gel tray with a well comb
    7. Let the agarose gel to sit at room temperature for 20-30mins until the gel is solid
    Sample Loading:
    1. Add (loading dye – purple or orange) around 2ul of the dye should suffice
    2. Once the gel has fully solidified place it in the box.
    3. Fill gel with 1xTAE until gel is submerged till the mark on the box.
    4. Load the Gene Ladder in the first lane of the gel
    5. It is recommended but not required to take a space lane where it is not filled in
    6. Run the gel at 80-150V until the process is approximately 75%-80% of the way down the gel, keep it running for 1-1.5hours
    7. Turn OFF the power, disconnect the electrodes and remove gel
    8. Using (MACHINE) with UV light device you are able to visualize the DNA fragments


    Miniprep Extraction


    Procedure - Retrieved from Qiagen Miniprep Kit

    1. Pellet by centrifuging for 3min at 3000rpm/ 6800g’s. Resuspend pellet bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
    2. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Mix gently by inverting the tube. Do not vortex, because this will result in shearing of genomic DNA and contamination of plasmid. Continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
    3. Add 350 μl Buffer N3. Mix immediately and thoroughly by inverting the tube 4–6 times. To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g., ≥5 ml) may require inverting up to 10 times. The solution should become cloudy.
    4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet will form.
    5. Apply 800 μl of the supernatant from step 4 to the QIAprep 2.0 Spin Column by pipetting.
    6. Centrifuge for 30–60 s. Discard the flow through.
    7. Recommended: Wash the QIAprep 2.0 Spin Column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow through. This step is necessary to remove trace nuclease activity when using endA+ strains, such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains, such as XL-1 Blue and DH5α, do not require this additional wash step.
    8. Wash QIAprep 2.0 Spin Column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
    9. Discard the flow through, and centrifuge at full speed for an additional 1 min to remove residual wash buffer. Important: Residual wash buffer will not be completely removed unless the flow through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
    10. Place the QIAprep 2.0 Spin Column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep 2.0 Spin Column, let stand for 1 min, and centrifuge for 1 min.


    Polymerase Chain Reaction


    Procedure




    Restriction Digestion


    Procedure 1 unit of restriction enzyme will completely digest 1ug of substrate DNA in a 50ul reaction in 60 minutes.

    For the Enzyme: Keep always on ice Last component to add Mix components by pipetting mixture up and down - flick reaction tube In general - recommended 5-10units of enzyme per ug DNA & 10-20 units for genomic DNA in a 1 hour digest

    DNA: Free of contaminants.

    Buffer: Use at 1x concentration.

    Reaction volume:

    References

      • Machado, L., Magnusson, M., Paul, N. A., Kinley, R., de Nys, R., & Tomkins, N. (2016). Identification of Bioactives from the Red Seaweed Asparagopsis taxiformis that Promote Anti-methanogenic Activity in vitro. Journal of Applied Phycology, 28(5), 3117–3126. doi:10.1007/s10811-016-0830-7
      • Roque, B. M., Brooke, C. G., Ladau, J., Polley, T., Marsh, L. J., Najafi, N., ... & Hess, M. (2019). Effect of the macroalgae Asparagopsis taxiformis on methane production and rumen microbiome assemblage. Animal Microbiome, 1(1), 1-14.
      • Thapa, H. R., & Agarwal, V. (2021). Obligate Brominating Enzymes Underlie Bromoform Production by Marine Cyanobacteria. Journal of Phycology.
      • Thapa, H. R., Lin, Z., Yi, D., Smith, J. E., Schmidt, E. W., & Agarwal, V. (2020). Genetic and Biochemical Reconstitution of Bromoform Biosynthesis in Asparagopsis Lends insights into Seaweed Reactive Oxygen Species Enzymology. ACS Chemical Biology, 15(6), 1662-1670.
      • Zhu, P., Li, D., Yang, Q., Su, P., Wang, H., Heimann, K., & Zhang, W. (2021). Commercial Cultivation, Industrial Application, and Potential Halocarbon Biosynthesis Pathway of Asparagopsis sp. Algal Research, 56, 102319. https://doi.org/10.1016/j.algal.2021.102319

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