Team:CSU CHINA/Notebook

Proof of Concept


Week 1: (16-22 August)

After having identified the components that needed to be transferred to the engineered algae, our first task was to build them into plasmids that could be transferred to the aggregates. We used the enzyme digestion method to attach the component fragments purchased from GenScript to the PVZ322 plasmid.

Week 2: (23-29 August)

To obtain a large amount of alga, 1ml of alga (OD=1) was pipetted into 40ml BG11 liquid medium in a 50ml centrifuge tube for propagation.

To determine whether the plasmid PVZ322 can be expressed in our synechocystis, we attempted to use natural transformation to transform the empty pVZ322 plasmid.

Synechocystis OD=0.8, 200μL
PVZ322 100ngμL, 50μL
Transformation Time 6h
Medium BG11, 1.5%Agar, sugar-free

The new algal fluid turned green after 12 days, and the result was: no algal blooms on the resistant BG11 plate, indicating that the algal transformation method or operation was wrong.

Week 3: (6-12 September)

In order to shorten the culture cycle of synechocystis, we pipetted algae seeds in the sugar-free BG11 liquid medium and 0.9% glucose BG11 liquid medium at a scale of 1:10.In order to eliminate the problem of conversion operation, we added parallel group, negative control and positive control.

The algal in sugar-free BG11 liquid medium turned green after 7 days, and the algal fluid in 0.9% glucose BG11 liquid medium turned green after 3 days, but one tube was infected with bacteria. The results showed that the sugar medium could shorten the culture period obviously, but increased the risk of bacterial infections.

Week 4: (13-19 September)

To successfully transform the plasmid PVZ322 into the synechocystis, we improved the transformation method and used the solid medium BG11 with sugar to culture and screen the transformed algae.

Synechocystis OD=0.8, 200μL
PVZ322 100ng/μL, 50μL
Transformation Time 6h
Medium BG11, 1.5%Agar, 0.9% sugar

To know exactly the growth period of wild-type synechocystis, we took samples every 1 day to detect the absorbance at 730nm.

0 0.754
29.5 1.052
48.5 1.298
71 1.4
130.5 2.09
185.5 2.45
209.5 2.47
233.2 2.51

The results showed that in our culture conditions, when the initial OD was 0.8, the synechocystis could be used for transformation for culturing about 30h.

Week 5: (20-26 September)

We continued to measure OD of several bottles of new synechocystis to obtain more accurate growth curve.

Since the last transformation was not successful, the method still needs to be improved.

Synechocystis OD=0.8, 400μL
PVZ322 100ng/μL, 100μL
Transformation Time 6h
Medium BG11, 1.5%Agar, 0.9% sugar

Week 6: (27 September-3 October)

We went on to determine the OD value of synechocystis in the previous week.

Since the last transformation was not successful, the method still needs to be improved:

Synechocystis OD=0.8, 400μL OD=0.8, 400μL
Mixed Cellulose Ester With Mixed Cellulose Ester Without Mixed Cellulose Ester
PVZ322 100ng/μL, 100μL 100ng/μL, 100μL
Transformation Time 6h 6h
Medium BG11, 1.5%Agar, 0.9% sugar BG11, 1.5%Agar, 0.9% sugar

Week 7: (4-10 October)

We attempted to test the cadmium tolerance curve of wild-type synechocystis.

Surprisingly, we stumbled upon the fact that the PVZ322 plasmid could not be transformed naturally, so trisophily conjugation was used.

Week 8: (11-17 October)


1. Test the availability of cadmium determination reagent kit

The sample order:A(1-6) standard sample in kit B(1-4) standard sample by me

1 2 3 4 5 6
A 0 0.4 1.2 4.7 10.4 32.8
B 1 1 1 1 \ \

2. Result of availability (availability test1):

Theoretical concentration:0.976179401

Excuse me? The curve is absolutely upside down, but I got the right concentration?

Do it again the other day! With a new kit! (this one must be polluted by my dear friends)


1. different densities of Synechocystis deal with cadmium

50mL / OD=0.17, 0.46, 0.5, 0.61, 0.78 / deal with cadmium of 0.1mg/L (density gradient test1)

50mL medium with 5μL of 1g/L Cd2+ stock

Take sample after 0.5h,1h,1.5h,2h,4h,12h

2. prepare new generation of Synechocystis for futher use (We are short of Synechocystis !)

3. prepare SA and CaCl2 stock----200mL

4. Immobilize Synechocystis experiment(MBs test1)

50 Ca-Alg-MBs, 50 Ca-Alg-MBs with Synechocystis

Take sample after 0.5h,1h,1.5h,2h,4h,12h>

5. Result of availability (availability test2)

Oh, my holy ****, why the curve is always, always upside down!

I need a new kit!


Neaten all the Synechocystis we have:





1. different densities of Synechocystis deal with cadmium

30mL / OD=0.42, 0.62, 0.89, 1.16, 1.34, 1.45 / deal with cadmium of 0.1mg/L (density gradient test2)

2. Synechocystis deal with different concentration of cadmium

30mL / Initial OD=0.7 / 0.01,0.1,0.5,1,2 mg/L Cd (Cd gradient test1)

3. gain sample of medium in (density gradient test1) and (MBs test1)

4.finally! a new kit!

What a beautiful curve!


Determination of the samples before

Emmm, why we get so awful results out of so beautiful standard curve

I guess the protein in the solution will affect the kit, the experiment operation is also important. Discard the data, try it again


Try to determine abnormal data gained in yesterday

Emmm, even worse.

Don’t want to do more experiment today.


Do every experiment once again

density gradient test3: OD=0.42, 0.62, 0.89, 1.16, 1.34 (0.1mg/L, 2mg/L)

Cd gradient test2: 0.01, 0.1, 0.5, 1, 2 (OD=0.7)

MBs test2: 30mL/ 50 MBs with PCC6803+50 MBs+ PCC6803 / 0.1mg/L+ 2mg/L Cd2+

Week 9: (18-25 October)


Gain the results seems to make sense(density gradient test3)