Team:ECUST China/Experiments
First of all, according to our experimental design, we need to transfer the total of four segments of exogenous gene. Because the single plasmid cannot carry all the exogenous gene, we need to build the double plasmid system, which contributes to insert the gene cpcE and cpcF encoding lyase into P426 SNR52p - gRNA plasmid and insert gene cpcA that encodes alpha subunit of phycocyanin and gene pcyA that encodes ferredoxin oxidoreductase into pTDH3 plasmid. After consulting with Professor Cai Menghao, an expert in the study of Saccharomyces cerevisiae in our school. We borrowed plasmids from Professor Cai Menghao's laboratory and after collaborated with Genscript Company. Genscript company helped us construct the exogenous gene into two plasmid skeletons and conducted sequence optimization. However, in order to make the plasmid more suitable for our experimental system, we constructed the expression box of the plasmid by ourselves. The experimental process is as follows:
To rebuild the plasmid , after getting the first edition of plasmid from company we finished storage，expansion and extraction of plasmid and so on. At the same time we still need to design primers, we chose the Primer Premier and Snapegene as Primer design software and by PCR primers calculator to calculate the parameters such as annealing temperature. After designing the primers, we obtained the small fragments we needed through PCR for splicing and reconstruction.
The target fragments were amplified by PCR using DNA polymerases Pfu, Taq or Prime Star, and the gel was recovered to measure the concentration of nucleic acid fragments.
PCR segments by gel extraction:
1-4: CYC1(terminator)
5-8: cpcE
9: GAP (promoter)
10-13: cpcF
14-17: cpcA
1-4: ADH1(Terminator)
1-4: LARGE fragment of P426
We carry out overlap according to the map of plasmid construction and gel recovery for corresponding fragments to measure the concentration of nucleic acid fragments. Overlap system (50 µ L) : Pfu enzyme, two segments to be connected (1:1 mole number), plus ddH2O Supplemented with 46 µ L system, 7-10 cycles were amplified, and then P1 and P4 primers were added to amplify 22-35 cycles.
1-4: GAP+cpcE+CYC1
5-7: GAP+cpcF
8: GAP+cpcA+ADH1
After the fragments have been connected, the plasmid was introduced into Escherichia coli (DH5α). 90 μ L of competent bacteria was added into each EP tube, 10 μ L of plasmid was added into Escherichia coli, evenly beaten, glycerol was added into 20 μ L.Place in ice for 1min and in heat engine at 42.1℃ for 1.5 to 2 minutes (transformation).Since both of our plasmids carry a gene screening marker for ampicillin resistance, the transformed E. coli is lined on a plate with ampicillin resistance. Single colonies were selected after 12 h culturing.
Meanwhile, plasmid was extracted from the colony. The colonies were subjected to PCR, with the PCR system as shown below. Finally, electrophoresis verification, glue recovery and Sanger sequencing were carried out.
1-24: cpcA_pTDH3
1-24: cpcE/F_p426
The gel of the 2 and 4 pore of CpcA_pTDH3 and the gel of 8,15 and 24 pore of the CpcE /F_p426 PCR systems (shown on the picture above)were sent for sequencing.
Sequencing result diagram:
Comparison of results determined by the company showed that our plasmid
construction results were consistent with our design expectations.
After the early stage of the plasmid construction has been completed, we will further introduce the dual-plasmid system into saccharomyces cerevisiae BY4741 which is the auxotroph stain of our target host saccharomyces cerevisiae S288C . The genotype for BY4741 is [genotype, Mata His3 Δ 1 Leu2 Δ 0 Met15 Δ 0 Ura3 Δ 0]) and there are Ura gene in plasmid P426 SNR52p - gRNA, Leu gene in plasmid pTDH3, from which we can carry out the selection of the successful transformed yeast using bideficiency medium.
General yeast medium (YPD or YEPD medium) : yeast extract 10g, protein 20g, glucose 20g, constant volume to 1L, 2% AGAR powder can be added to become a fixed medium.Sterilization temperature: 115℃.
Saccharomyces cerevisiae screening medium (SD-URA /Leu medium) : YNB medium dry powder 6.7%.Both Met and His mother solution were prepared 20mg/ mL with 250 µ l of mother solution per 100mL medium.Sterilization temperature: 115℃.
1. Put the micropluser cuvettes in the super clean bench in advance and dry
it for 3-4 hours.
2. Remove the competent cell from -80C refrigerator and melt them in palm. The
80ul competent cells and about 1ug DNA samples were mixed and transferred to a 0.2cm pre-frozen
micropluser cuvettes for ice bath for 5 min.
3. Put the micropluser cuvettes into the chute of
the rotary instrument set in Pic "mode, shock once, then quickly add 1.0m1 pre-cooled 1Mo1 /L
sorbitol solution to the micropluser cuvettes, gently blow and mix, and transfer 500U of bacterial
liquid from the micropluser cuvettes to EP tube with 500 m YPD liquid medium, and culture for about
2h on a shaking bed at 30℃ and 200 r/min.
4. Absorb an appropriate amount of the bacterial liquid
recovered from the electrical transfer in step (3), spread it on the corresponding screening plate,
and incubate it in an inverted dry incubator at 30℃ for about 2 days until the transformants visible
to the naked eye grow.
After successfully transforming the double plasmid into yeast, we verified phycocyanin by SDS. After reviewing the data, we found that the molecular weight of phycocyanin was 19.7KD, and we analyze the results according to the bands.
SDS PAGE Protocol:
1. Make the separating gel and stacking gel.
2. Pour the running buffer (electrophoresis buffer) into the inner chamber and keep pouring after overflow untill the buffer surface reaches the required level in the outer chamber.
3. Prepare the samples.Mix your samples with sample buffer (loading buffer).Heat them in boiling water for 5-10 min.
4. Load prepared samples into wells and make sure not to overflow. Loading protein marker into the first lane. Then cover the top and connect the anodes.
5. Set a volt and run the electrophoresis when everything's done.Generally, about 1 hour for a 120V voltage and a 12% separating gel.
6. Immerse the gel with staining solution,and slowly shake it on horizontal rotator for about 20-30min.
7. Immerse the gel in destaining solution and put it on the same shaker for about 20-30 mins.Change the destaining solution for 3-5 times untill you can see clear bands with almost no blue background.
8. 100ml fixing solution : 50 % Methanol, 10% Acidic Acid, 50 µl Formalaldehyde for one hour to overnight.
9. Wash gel with 50 % ethanol for three times (no less than 20 minutes each).
10.Treat gel for 1min with hypo solution (Sodium Thiosulfate solution 20 mg/100 mL). SAVE 2 ml FOR LATER STEP.Over treating with the hypo solution will result in darker gel in the end.
11. Wash with water for three times (20 seconds each).
12. Treat gel with Silver Nitrite solution (200 mg/100 mL) for 30 minutes.Over treating will result in unwanted artifacts in final stage.
13. Wash with water for three times (20 seconds each).
14. Develop gel in 100 mL developing solution (6 g Sodium Carbonate, 2 mL of the hypo solution, 50 µl Formalaldehyde). This step can take from one minute to 30 minutes.
15.Once developed, stop with 5% Acidic Acid. Once We saw the bands, stopped developing the gel immediately.Store gel in fixing solution.
We carry out color-rendering experiment to provide basic data for the modeling group, and these data provide support for the construction of color-rendering model.
Experimental steps:
1. 325 mg/L phycocyanin mother solution 20 mL was prepared
2. Phycocyanin solutions with different concentrations were prepared according to the table below, and 3 centrifuges were prepared for each concentration, which were evenly divided into groups A, B and C
3. Three sets of centrifuge tubes A, B and C were immersed in constant temperature water bath at 30℃, 40℃ and 50℃ for 40min respectively
4. Remove the centrifuge tube and shake for 3 s
5. Take 20 μL solution from each centrifuge tube to the plate, add equal volume of deionized water to the first well in each row as blank control, and measure the absorbance value of the solution at 614 nm wavelength with a microplate reader
6. The measured absorbance value minus the absorbance value of the peer deionized water is the final absorbance value of phycyanin solution
Experimental results:
GET IN TOUCH
+86 15821261548
IGEM_ECUST@outlook.com
ID:IGEM_ECUST
ECUST_China
East China University of Science and Technology
Shanghai,China
