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: