Team:UChile OpenBio-CeBiB/Safety/LabProtocols

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Experiments

Chlamydomonas reinhardtii growth

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.

[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)


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]
$$K_2 HPO_{4}$$ 10.8
$$KH_2 PO_4$$ 5.6

Table 3: Solution A (for 500 ml)
Compound Mass [gr]
$$KH_4 Cl$$ 20
$$MgSO_4\cdot7H_2O$$ 5
$$CaCl_2\cdot2H_2O$$ 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.

    Sequences

  • Design of the sequence of the fusion protein FBP/SBPase-GFP (cTP-FBP/SBPase-GFP and cTP-GFP-FBP/SBPase) is in Annex. Moreover, the control protein will be cTP-FBP/SBPase. The sequence will be obtained by DNA printing.
  • Design of the sequence of B12 responsive element-RFP is available in Annex (MetE-RFP operator). It will be obtained by DNA printing. The sequences cTP-FBP/SBPase, cTP-FBP/SBPase-GFP, cTP-GFP-FBP/SBPase and METE-RFP operator will be adapted to the Codon bias of Chlamydomonas reinhardtii by the page https://www.idtdna.com/CodonOpt.
  • Design of the parts, incorporation of prefixes and suffixes. Prefix: ExoRI, NotI, Xbal. Suffix: SpeI, NotI, PstI. These are included in the BioBrick of the form:

    • 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 little plates: LB agar 15 ml each approx + ampicillin. The plates are prepared in the laminar flow hood.

    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 centrifuge (tube 1)

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

    Plate 3: centrifuge tube 2. Peller with some of 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 ambient 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.