Protocol

Algae transformation

BG11 medium

The content of mother solution

Mother liquors	substance	content(g/L)	For 100mL （g）
1	K₂HPO₄·3H₂O	40	4
2	MgSO₄·7H₂O	75	7.5
3	Ferric ammonium citrate	6	0.6
3	Citrate acid	6	0.6
4	CaCl₂·2H₂O	36	3.6
5	EDTA-Na2	1	0.1
6	H₃BO₃	2.86	0.286
	MnCl₂·4H₂O	1.81	0.181
	ZnSO₄·7H₂O	0.222	0.0222
	NaMoO₄·5H₂O	0.39	0.039
	CuSO₄·5H₂O	0.079	0.0079
	Co（NO₃）₂·6H₂O	0.04	0.004

Liquid medium (for 1L)

1ml of each mother liquors, besides, add

NaNO₃	1.5g
Na₂CO₃	0.02g

Adjust pH to 7.5-7.55, 121℃ autoclaved for 20mins.

Solid medium (for 1L)

1ml of each mother liquors, besides, add

NaNO₃	1.5g
Na₂CO₃	0.02g
Na₂S₂O₃	3g
TES	2.22
Agar	1.5% Which means 15g

Adjust pH to 7.5-7.55,121℃ autoclaved for 20mins. Prepare solid plate before use. Be sure the antibiotic is active and in appropriate concentration.

Transformation of hunger

Time	Steps
Day 1 at noon	Set the culture，the initial OD730 is 0.1
Day 2 in the evening	After 1.5 days，the OD730 would approach to about 0.5（even though 0.3-0.7 is ok）.
	Centrifuge at RT3000g10min，remove the supernatant，add 1ml liquid medium，measure the od730 by nanodrop, then calculate the volume of cyanobacteria to adjust the ultimate od730 to 2.5.
	Calculate the volume of DNA（5-10ug，usually take 8ug）
	Mix the DNA, cyanobacteria and liquid medium in a 15 ml centrifuge tube to make the total volume is 500ul and the od730 is about 2.5, set a negative control (without DNA).
	Overnight and then shake slightly by hand for about 4 times.（every 2-3 hours）
Day 3 at afternoon	Take 200ul culture, spread it on solid plate with suitable antibiotic, put the plate into illumination incubator at 30℃.

Then wait for several days (depends on the gene)
If the solid plate with DNA have colony while the negative plate is clean, then we may conclude the natural transformation is successful, but we need three experiments for further determination.

1.

Pick up the colony，spread it in a solid plate (with antibiotic) in parallel, put it in illumination incubator to store the colony. (Not all the colony can grow because this can work as segregation.)

2

Pick up the colony

Set a 500ul liquid culture, then add it into an 100ml flask for further segregation.

Spread it on solid plate in parallel to store the cyanobacteria.

Set a 50ul liquid culture to do colony PCR

Colony PCR：put the 50ul liquid culture into liquid N2, then into 80℃ water, repeat these steps for another 2 times, take 0.5ul to do colony PCR.

PCR system：

2*Taq enzyme mix	5ul
Primer	0.5ul*2
Template（the liquid culture）	0.5ul
ddH₂O	3.5ul

PCR program：

95℃	3-4min
95℃	30s	35 times
60℃（ADS, the temperature depends on your primer）	30s
72℃（for taq enzyme）	2min（depends on the length of your gene）
72℃	5min
4℃	Forever

Run gel to determination.

Transformation of electricity

Day 1 at noon	Set the culture.
Day 1 at noon	Prepare the electric switch and electric tubes, wash the tubes with ddH₂O for several times, then wash with 75% ethanol, put them in a beaker, covering with 75% ethanol.
Day 3 in the evening or at the afternoon	Take out the electric tubes from ethanol, put them into 60℃ oven for at least 2 hours to dry the tube, sterilize them by UV before electrotransformation.
	Centrifuge at 8000rpm*5min (the OD730 is about 0.4), dilute the cell with sterilized ddH₂O (40,30,30,20,20ml separately) wash it for 5 times. (suggest 7 times at first time)
	Dilute the cyanobacteria with 200ul sterilized ddH₂O, mix it with DNA (at least 500ng) to make the total volume to 50ul，add them into electric tube, tunk for several times so that the liquid can reach to the bottom.
	Put the electric tube in electric switch correctly, choose Ec1 program, press “pulse”, wait for a moment, check the voltage and time. (The best condition is 12kV & 5ms, but we take 18kV & 4-6ms as well.) Add 1ml liquid BG11 culture immediately, mix and add back to 50ml BG11 culture, put it in shaking illumination incubator at 30℃*130rpm for 1day.
Day 4 in the evening	Collect the cyanobacteria at 8000rpm*5min, dilute the cell with 400ul liquid culture, mixed with 5ml soft agar BG11 medium, spread on solid medium (antibiotic added), put the plate into illumination incubator at 30 ℃.

Wait for several days (depends on your gene), then test your colony just like natural transformation.

Prove transformation success

Three experiments to prove the success of transformation

1.