Team:UChile OpenBio-CeBiB/Safety/LabProtocols

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Lab Protocols

The strain that will be utilized is cell wall deficient, the used in [1] is the strain cw15-30-derived UVM4 C. The culture medium in which they grow are TAP (solid) and TAPS (liquid). Light stimulation will be required, for which a structure capable of covering the Erlenmeyer flasks or the petri dishes in which C. reinhardtii would be growing while uniformly illuminating with the whole visible light spectrum will be designed and built. In [1] the lamp that is put into use has the following characteristics: Nano Light, 11 Watts, Dennerle, Vinningen, Germany, which provided constant light with the following details: 2500 lux, eq. 72.5 μE/m2∙s1.

For the construction of calibration curves the medium to be used is TAPS without acetate.


Table 1: Stock Solution for 1 L of TAP medium (adjust final pH to 7)
Solution/Compound Volume [ml]
1M Tris base (e.g. Trizma) 20
Phosphate Buffer II* 1
Solution A 10
Hutner's trace elements* 1
Glacial Acetic Acid 1

Table 2: Phophate Buffer II (for 100 ml)
Compound Mass [gr]
Dipotassium phosphate 10.8
Monopotassium phosphate 5.6

Table 3: Solution A (for 500 ml)
Compound Mass [gr]
Amonium chloride 20
Magnesium sulfide heptahydrate 5
Calcium chloride dihydrate 2.5

Regarding Hutner´s trace elements:

  • Mix all solutions except EDTA. Bring to boil, then add the EDTA solution. The mixture must turn green. When all is dissolved, cool to 70°C.
  • Keep temperature at 70°C, add 85 ml of hot KOH (20%).
  • Cool to room temperature and bring to 1 liter of final volume. -Generally, the solution will be initially green, but it will turn dark red or purple in the next days and will leave a redish brown precipitate. If no precipitate is formed or the solution remains green, check the pH. It must be around 6.7. If it is radically deactivated, try adding KOH or HCl to adjust it.
  • The procedure requires to cover the flask with a cotton plug (to allow air exchange) and swhirl it once a day for 1 to 2 weeks. Or even better, use a 5 or 10 ml pipette (with a cotton filter on top) pushed through a foam plug and inject air through the solution for a week.
  • Filter through two layers of filter paper Whatman #1. Repeat if necessary until the solution is clear.
  • The final product should turn close to a burgundy color. However, even a subtle change in pH can cause a color shift.
  • Store refrigerated or frozen in convenient aliquots.
  • This can be done in bigger batches – 2 or 3 liters, if it is preferred.
  • Source: http://www.chlamycollection.org/methods/media-recipes/hutners-trace-elements/

    TAPS

    It’s TAP medium supplemented with 1% w/v sorbitol.


    [1] Chávez, M. N., Schenck, T. L., Hopfner, U., Centeno-Cerdas, C., Somlai-Schweiger, I., Schwarz, C., ... & Nickelsen, J. (2016). Towards autotrophic tissue engineering: photosynthetic gene therapy for regeneration. Biomaterials, 75, 25-36.

    TAP (Tris-Acetate-Phosphate)




    Strain: chemocompetent top 10

    -20 minutes in the cold on ice within the EPS packaging + 1 min at 42° + 3 min on ice and the it was left at 37°C for 1h 30m.

    -Culture medium in eppendorf: TB

    -Medium in small plates: LB agar 15 ml each approx + ampicillin. The plates are prepared in the laminar flow cabinet.

    -All the procedure is made under burner (sterility)

    -Tube 1: TB + X colis + 4 ul o vector

    -Tube 2: TB + X colis + 1 ul < X < 4 ul of vector

    -Plate 1: 100 uL of E. coli without centrifugation (tube 1)

    -Plate 2: centrifuge tube 1. Pellet with some of the medium

    -Plate 3: centrifuge tube 2. Peller with some of the medium

    -After that, everything was left in the stove at 37°C

    -If grass is formed there would be needed to scratch and “dilute” and replate.

    -The rest of the vector is stored at -20ºC

    -Next day 2 colonies are inoculated from one of the plates, in 2 falcon tubes, respectively. These Falcon tubes contained 10-15 mL of liquid LB and ampicillin



    -First, two cultures with E. coli were taken, 2 mL were transferred in Eppendorf tubes, in duplicate (for a total of 4 samples)

    -A centrifugation at 8000 rpm was made for 2 m at room temperature.

    -After that, the process of cell resuspension, lysis and neutralization was initiated. For this, 250 uL of resuspension solution was added to each tube, and they were vortexed until homogeneous solutions were formed.

    -Then, 250 uL of lysis solution were incorporated and the tubes were carefully flipped around 10 or 12 times (A whiter coloration was visualized) Then, 350 uL of neutralizing solution were added, and the tubes were flipped again around 10 or 12 times (the formation of a viscous white substance was spotted) After that, it got carried to centrifuge at 8000 rpm for 5 minutes.

    -After the centrifugation, the supernatant was removed and transferred to the Thermo Scientific GeneJET Spin Columns, for later centrifuge at 8000 rpm for about 1 minute.

    -It is continued with the third step, of column washing: 500 uL of washing solution were added and it was centrifuged for 1 minute at 8000 rpm and then what was filtered was thrown away. Then, the empty column was centrifuged at 8000 rpm for 1 minute.

    -Finally, fourth step was carried out. The column was transferred in a new Eppendorf and 50uL of Elution buffer was added to the column. It was incubated for 2 minutes. Immediately, it was centrifuged at 14000 rpm for 2 minutes and obtain volume was recovered in a new Eppendorf.

    -200uL of plasmid solution was obtained, and they were distributed in 4 Eppendorfs of 50uL. To each one, 2uL was extracted for plasmids quantification, remaining 48 uL in each Eppendorf.

    -Obtained concentrations were:

  • 266,21 [ng/uL]
  • 290,02 [ng/uL]
  • 523,49 [ng/uL]
  • 509,38 [ng/uL]
  • For guarantee a bacterial culture stock, 700uL was extracted of E. Coli original samples and was mixed with 300 uL of glycerol in a Falcon tubes. This step was made to each Falcon tube and two preserved cultures of 70% E. Coli and 30% glycerol was made. The 4 Falcons with plasmid and two with E. coli were frozen at -20°C.



    We worked under laminar flow, this way it avoided that the external air enters. It had a HEPA filter which cleaned the air that was in contact with the samples.

    Despite previous conditions, the work surface and all the instruments that was used (matraces, Falcons, etc) had to be cleaned with ethanol

    -First, we had to count the cells from the culture of microalgae to be sure that the transformation was successful, because the sample that was subsequently be used could've had too much cell debris.

    -For the count, we used a Neubauer chember. For that, in a sterile Eppendorf tube and a sterile serological pipette, 15 mL were taken from the culture.

    -From the 15 mL, 100 uL were taken and were added to the chamber distributed in the two fronts. The objective was fill with the sample the zone chamber grid. The characteristics of the chamber were: the squares #4 were the smallest (inside the squares #3). In those we did cell count, obtaining a mean of 9 squares type 4, so:


    Figure I: Square

    Each square type 4 has an area of 0,0025 mm2, (indicated in the chamber's instructions) and each square type 3 had an area of 0,2 mm x 0,2 mm , equivalent to 0,04 mm2.

    The volume was calculated as V = 0,04 mm2∙0,1 mm = 4∙1e-3 mm3 = 4∙e-6 ml

    Using an average of 70 cells

    -17,5⋅e6 cells/mL were counted. According to the protocol, we need at least 3⋅107 célls/mL to make the transformation. Doing a little bit of calculations, we determined that we had to use 1,7 ml

    -On the other hand, in two eppendorf tubes that contained sterile water, 300 g of glass beads were added to each one, with a small pipette (before adding the glass beads, they must be treated with HCl the night before, then cleaned and finally autoclaved)

    -The beads in water allowed us to use them easier (then the water was eliminated)

    -Later, 1,7 mL from the culture must be added in each Eppendorf

    -The microalgae were centrifuged a 600 g for 5 min. The centrifugation aimed to create the pellet without the medium in which they are growing (TAPS medium)

    -Once obtained the pellet of C. reinhardtii, the supernatants were removed from each Eppendorf tube with a micropipette with filter tips to avoid contamination

    -The cells were resuspended in 300 uL of TAPS to each Eppendorf

    -Then, we took 5-7 ug of DNA (pBC1-CrGFP plasmid). The plasmid had a concentration of 0,5 ug/mL. Each Eppendorf had 5 ug of plasmid, 10 uL and had been taken .

    -The cells were mixed with the beads and water. The mix was homogenized in a vortex shaker during 20 s. Then, we waited a few minutes until the glass beads fell to the bottom.

    -The cells from the upper side were taken from the tubes (without beads), as much as possible. It is not relevant if some glass beads are taken with the micropipette.

    -Later, we added 5 mL of liquid TAPS medium to a Falcon of 15 mL.

    -In each Falcon 200 uL of C. reinhardtii were added.

    -The Falcon were labeled as “C.R. UVM4 pBC1 13/07/17”

    -This Falcon were wrapped in aluminum and left in dark all night, at a shaker at room temperature.

    NOTE: The cultures may last one week, then they must be refreshed.



    -The Falcon tubes of 50 mL were centrifuged at 600 g for 5 min.

    -They were centrifuged again at 850 g for 5 min, because a concentrated pellet must be formed, which was not accomplished in the last step.

    -The supernatant was eliminated.

    -100 uL of pellet were taken with a pipette with filter tips and were plated in Petri plates with TAPS growth medium with paromomycin to create a gel in the bottom.

    -The way to correctly plate is spreading the sample with the pipette over all the Petri plate.

    -This was done for the 2 Petri plates, that were labeled “C.R. UVM4 pBC1-GFP 14-09-17 (1 , 2)”.

    -The Petri plates were sealed with parafilm by the edge so that the liquid on the plaque could be dried. The next day, the plates had to be flipped (the agar in the upper side) to avoid the contamination by fungus.

    This transformation was a test, for when we have the vector ligated with the sequences which contains the gene that encodes FBP/SBPasa. The results of this transformation were negatives, the cultures did not grew. This can be explained because the Chlamydomonas r. strain used had already the vector.



    1.- Centrifuge the tube for 3-5s and 300 g as minimal, to the material contained not stay in the upside of the tube.

    2.- Add TE to obtained a final concentration of 10 ng/mL.

    3.- Put in a vortex mixer briefly.

    4.- Incubate for 20 min at 50°C

    5.- Put in a vortex mixer briefly and centrifuge again with the same conditions in 1.



    I. Preparation of the primers: in iceII.

    1.- Resuspension: preparation of a stock with 100 uM

  • Must be registered the quantity of [nmoles] labeled in each tube.

  • Must be added a volume of water equals a [nmoles x 10].

  • Example: Primer with 29.8 [nmoles], so, must be added 298 [uL] of water to obtain a stock concentration of 100 [uM].

  • Put in a vortex mixer and centrifuge.

  • 2. Prepare a solution with 5 [uM]

  • In a tube of 0,6 mL put 95 [uL] of sterile and nuclease free water.

  • Mix 5 [uL] of the primer stock at 100 μM.

  • Put in a vortex mixer and centrifuge.

  • II. Preparation of a dNTPs stock at 2 nM.

  • Put 92 uL of sterile water free nuclease in a tube of 0,2 mL

  • Add 2uL of dATP 100 uM

  • Add 2uL of dDTP 100 uM

  • Add 2 uL of dGTP 100 uM

  • Add 2 uL of dCTP 100 uM

  • III. PCR component by reaction

  • 5X Phusion HF o GC Buffer 10 µL

  • 2 mM dNTP 5µL

  • 5 uM FW primer 5µL

  • 5 uM RW primer 5µL

  • Template DNA (gBlocks) 1µL

  • Phusion polymerase 0,5µL

  • Nuclease Free Water 23,5 µL

  • TOTAL 50 µL

  • Program of 30 cycles:

    1) Start at 94°C for 2 min

    2) 30 ciycles

  • Denature at 94°C for 15 sec
  • Anneal at 65,2°C for 35 sec
  • Extension at 72°C for 50 sec (it depends on the sizes of the sequences; approx 20 sec per 1000 bp)
  • 3) Final Extension at 72°C for 5mn



    Was prepared an agarose gel to extract the DNA amplified in the experimental session did previously. Was charged the 5 sequences plus a ladder to have a comparison point with the molecular mass of the obtained bands.

    Once obtained the gel, observe it under UV light. Were done splits of the bands corresponding to the amplified DNA. In all the cases, except for Seq 4 and Est 2, can be watched very sharp fluorescents bands which correspond the amplified DNA. For Seq 4 and Est 2 can be watched two fluorescents bands very close that could be the corresponding amplified DNA, but according to their molecular mass (1.405 kb for Seq 4 and 665 pb for Est 2) they may were identified and may were cut those bands from the gel.

    Then, we made a purification of each DNA sample, according to the protocol for the extraction from the agarose gels of Sigma-Aldrich, that is detailed next:

    1.- Weigh the gel slice in a tared colorless tube. Add 3 gel volumes of the Gel Solubilization Solution to the gel slice. In other words, for every 100 mg of agarose gel, add 300 mL of Gel Solubilization Solution. Incubate the gel mixture at 50-60 °C for 10 minutes, or until the gel slice is completely dissolved. Vortex briefly every 2-3 minutes during incubation to help dissolve the gel.

    2.- Prepare binding column. Preparation of the binding column can be completed while the agarose is being solubilized. Place the GenElute Binding Column G into one of the provided 2 ml collection tubes. Add 500 mL of the Column Preparation Solution to each binding column. Centrifuge for 1 minute. Discard flowthrough liquid.

    3.- Add isopropanol. Add 1 gel volume of 100% isopropanol and mix until homogenous.

    4.- Bind DNA. Load the solubilized gel solution mixture into the binding column that is assembled in a 2 ml collection tube. Centrifuge for 1 minute after loading the column each time. Discard the flow throughliquid.

    5- Add 700 mL of Wash Solution (diluted from Wash Solution Concentrate G as described under Preparation Instructions) to the binding column. Centrifuge for 1 minute. Remove the binding column from the collection tube and discard the flow-through liquid. Place the binding column back into the collection tube and centrifuge again for 1 minute without any additional wash solution to remove excess ethanol. Residual Wash Solution will not be completely removed unless the flow-through is discarded before the final centrifugation.

    6.- Elute DNA. Transfer the binding column to a fresh collection tube. Add 50 mL of Elution Solution to the center of the membrane and incubate for 1 minute. Centrifuge for 1 minute.


    Table 1: Volumes of each material
    DNA Sample Endonucleases Endonuclease Volume [ml] Buffer Volume NB 3.1 [uL] Dna Volume [uL] Water Volume [uL] Final Volume [uL]
    Est_2 EcoRI and PstI 1 EcoRI - 0.5 PstI 2 10 6.5 20
    Est1_2 EcoRI and PstI 1 EcoRI - 0.5 PstI 2 10 6.5 20
    Seq1_2 EcoRI and NdeI 1 EcoRI - 0.5 NdeI 2 10 6.5 20
    Seq2_2 EcoRI and NdeI 1 EcoRI - 0.5 NdeI 2 11 5.5 20
    Seq4 NotI and XhoI 0.5 NotI - 0.5 XhoI 2 12 4.5 20
    pSB1A3 (2 samples: for Est1_2 and Est2 sequences) EcoRI and PstI 1 EcoRI - 0.5 PstI 2 1 15.5 20
    pBC1 (2 samples: for Seq1_2 and Seq2_2 sequences) EcoRI and NdeI 1 EcoRI, 0.5 NdeI 2 1 15.5 20
    pBC1 (for Seq 4 sequence) NotI and XhoI 0.5 NotI, 0.5 XhoI 2 1 15.5 20


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