Team:ECUST China/Proof Of Concept
According to the biosynthesis pathway of phycocyanin α subunit, we use PCR to clone the four genes we need, phycocyanin apoprotein (cpcA), ferredoxin oxidoreductase (pcyA), phycocyanin lyase α subunit (cpcE) and phycocyanin lyase β subunit (cpcF). These four genes were transformed into PTDH3 plasmid and p426-SNR52p plasmid, forming our dual-plasmid system. See how the four genes and their expression boxes were correctly inserted into the vector. Note that the pcyA gene’s expression box was inserted by the company correctly, so its correctness was only confirmed in subsequent sequencing.
PCR recycling fragment
1-4: CYC1(terminator)
5-8: cpcE
9: GAP (promoter)
10-13: cpcF
14-17: cpcA
After the construction of two plasmid , we transferred the dual-plasmid into Escherichia coli DH5α for amplification. Because both of our plasmids carried ampicillin resistance gene so we smeared the transformed E.coli on the plate with ampicillin to select the interest E.coli. Single colonies were selected to be further cultivated in shacking culture after 12 hour’s growth.
At the same time, we extracted plasmids from the E.coli colony. And the purity of the two plasmids we extracted was high, their OD260/OD280 values were 1.798 and 1.804 respectively. We firstly use PCR and then electrophoresis to vertify the correctness of our plasmid, and then we recover the gel and send the plasmids to company, using Sanger Sequencing to further vertify the correctness of our plasmid.
1-24: cpcA_pTDH3
1-24: cpcE/F_p426
CpcA_pTDH3 took 2 and 4 PCR systems and bacterial solution and sent them for sequencing cpcE /F_p426 PCR systems 8, 15 and 24 were taken and sequenced
Results:
Using our two plasmids as a template, PCR detection showed that the fragment size
was consistent with the corresponding gene, and the company sequencing confirmed that the four genes
and their expression boxes connected to the double plasmid system were splicing correctly.
In the synthetic pathway we constructed, the intermediates 3Z-Phycocyanobilin and Phycocyanin α subunits will only appear in yeast on the premise of correct expression of the pathway. So our primary goal is to detect the presence of these two substances.
In the detection of the presence of phycocyanin lyase (cpcE and cpcF encoded), we focused on the reaction process itself. The alpha subunit of phycocyanin consists of a polypeptide chain of the cpcA encoded protein and a tetrapyrrole structure of 3Z-Phycocyanobilin (PCB) molecules. Normally, the alpha subunit connect with one 3Z-Phycocyanobilin, which is covalently crosslinked with the cysteine residue in the polypeptide chain via thioether bond. Only in the circumstance that 3Z-Phycocyanobilin (PCB) was catalyzed by lyase to bound to the appropriate site of phycocyanin α subunit, α -Cys-84, can the phycocyanin formed. Therefore, as long as we detect the presence of the final product, we can simultaneously determine the presence of the product lyase(cpcE and cpcF encoded).
The green base is α-phycocyanin. The purple ligand is 3Z-phycocyanobilin
There is no phycocyanin in wild type S. cerevisiae S288C, so we cultured the transformed s. cerevisiae and the control yeast without the transformed dual-plasmid at the same time. When the protein was expressed in large quantities in logarithmic growth phase, the two lines of the yeast we cultured were ultrasonically broken and then we use SDS-PAGE analysis to compare the two lines.
We found in EMBL-EBI that the molecular weight of Phycocyanin α subunit encoded by cpcA is 17587 Da, but we also searched a large number of other literatures. The molecular weight of the α subunit of phycocyanin is 17.6 k Da[10],and it has also been reported as 16.6 k Da[9] or 21k Da[1]. Therefore, our results can be correct when the comparison bands appear in the range of 16~22KD.
SDS-PAGE results showed that there was an obvious blot about 20kD in the phycocyanin expressed s. cerevisiae, while there was no blot in the control group.
The principle of the pigment protein zinc electrophoresis is that pigment can form a chelate with divalent zinc ions and the chelate can emit fluorescence under a certain wavelength of exciting light. The cells of the recombinant system were ultrasonically broken, and the supernatant was taken after centrifugation. Then the purified protein was prepared. The final concentration of 10% trichloroacetic acid was added and placed at -20℃ for 15 min to precipitate the purified protein. The precipitate was washed with acetone for 3 times, dried, and then added with sample loading buffer and mercaptoethanol (9:1), boiled at 100℃ for 5 min, centrifuged, and the same amount of supernatant was taken from each sample for SDS-PAGE. After electrophoresis, the gel was soaked in 1.5 mol/L zinc acetate solution for 15 min at room temperature and detected under 280 nm UV lamp. After soaking in zinc ion solution, the recombinant protein showed orange fluorescence under ultraviolet light excitation.
This could prove the successful covalent connection between the protein and pigment in S. cerevisiae.
Phycyanin has a characteristic absorption spectrum. Phycocyanin α subunit’s characteristic atabsorption spectrum is at 620nm and it’s maximum emission wavelength is 640.8 nm. Full-wave microplate tester was used for detection, the scanning range of absorption spectrum was 500-800 nm and steps were set to 10nm. The results are as follows:
In order to make our results more accurate, we narrowed the condition range. The scanning range of absorption spectrum was 590~640 nm, and the steps were set to 5nm, and the results were as follows:
The results showed that the maximum absorption wavelength was 620nm, which further suggested that the recombinant protein was phycyanin α subunit. It can also demonstrate that 3Z-Phycocyanobilin (cpcA encoded) with tetrapyrrole structure was catalyzed by lyase (cpcE and cpcF encoded) to bind with phycyanin α subunit, successfully forming our final product, phycocyanin.
Reference：
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[5] Ming H L , Castillo G , Ochoa-Becerra M A , et al. Phycocyanin and phycoerythrin: Strategies to improve production yield and chemical stability[J]. Algal Research, 2019, 42:101600-.
[6] Liang Y , Kaczmarek M B , Kasprzak A K , et al. Thermosynechococcaceae as a source of thermostable C-phycocyanins:properties and molecular insights[J]. Algal Research, 2018, 35:223-235.
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[8] 2018.Lee S. Ifhar, Dorit Ben-Shachar,Chapter 8 - Heme metabolism, mitochondria, and complex I in neuropsychiatric disorders,Editor(s): Illana Gozes, Joseph Levine,Neuroprotection in Autism, Schizophrenia and Alzheimer's Disease,Academic Press,2020,Pages 173-207,ISBN 9780128140376,
[9] 莫柳达. 螺旋藻中藻蓝蛋白的分离纯化、荧光探针研制及其在快速检测中的应用[D].福州大学,2018.
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Only when the product have been used in the downstream industry and be proved to be feasible and superior than the original old method, can our project truly been proved to be valuable and troubleshooting.
After researched the references, we found yeast mud produced in the last step of beer fermentation was usually wasted and the lengthy extraction steps to obtain phycocyanin from algae wasted a lot of unnecessary energy. Combining the characteristic of our modified yeast, we build the Phycocyanin Beer Production Factory to solve the two problems mentioned above simultaneously.
The following picture shows how we design the process of factory:
In the fermentation of beer, our modified yeast faces two main obstacles. One is that the fermentation environment for our yeast to produce phycocyanin is aerobic while the environment for beer fermentation is anaerobic. The second problem is that phycocyanin is expressed in the cytosol of yeast while the last step for beer fermentation involves filtration to remove the yeast which is important to ensure the taste of beer. We use two-stage fermentation to tackle with the first problem, solving the difficulties that the culture medium and fermentation environment is different for expressing phycocyanin and beer production. And we target self autolysis as the key to solve the second problem. Through controling the PH, we can control the degree of self autolysis which will release intracelluar molecules in the last step of fermentation, thus letting the beer turn blue without effecting the taste of it.
It is worth to mention that our factory carries on the innovation in term of recycling yeast slurry after beer fermentation to gain pure phycocyanin and at the same time produce phycocyanin beer to maximize the profits and reduce the environmental pollution that may cause from the extraction step to produce phycocyanin from algae.
After consulting Pro. Wang Xuedong, he convinced of our Phycocyanin Beer Production Factory idea. So we further researched the detail data for fermentation such as the temperature , pH ,period and so on, we proceed to further complete our factory through 3D modeling. The following picture shows our 3D model we constructed:
Besides process diagram and 3D modeling, we also paid attention to the total cost of the factory which is vital for a new factory to be implemented in the real world. (See more in our hardware web-page.) After rough estimated, we find that our factory is economical, not to say it won’t cause environment pollution compared to the traditional way to produce phycocyanin and treat with yeast mud.
Based on this hardware, we can later magnify the production scale with increased size and parallel number of bioreactors. The factory is characterized with our own yeast’s feature and can really make our concept of ‘Magic Blue’ come true!
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IGEM_ECUST@outlook.com
ID:IGEM_ECUST
ECUST_China
East China University of Science and Technology
Shanghai,China
