Team:CSU CHINA/Notebook
Notebook
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.
| TIME(H) | OD730 |
|---|---|
| 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)
10.12.
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)
10.13
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!
10.14
Neaten all the Synechocystis we have:
OD=0.9----100mL
OD=0.5----50mL
OD=1.2----50mL
OD=1.0----50mL
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!
10.15
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
10.16
Try to determine abnormal data gained in yesterday
Emmm, even worse.
Don’t want to do more experiment today.
10.17
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)
10.18
Gain the results seems to make sense(density gradient test3)