Team:Mingdao/Demonstrate

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JUDGING FORM ⇗
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Biological Function

Biobrick Construction

Gene Expression & Purification

CA

CYP2E1

Discussion

To increase our algae system's efficiency of reducing indoor pollutants such as CO2 and VOCs, we applied two enzymes, carbonic anhydrase(CA) and cytochrome P450 2E1(CYP2E1) to improve algal absorption of CO2 and VOCs, respectively. We produced enzymes by engineering Bacillus subtilis 168 with BioBricks we created and then finally put them into our system. We also did some experiments to analyze the activity of our enzymes and test whether our system is efficient enough to refresh indoor air.

BIOLOGICAL FUNCTION

Carbonic anhydrase (CA)

CA is an enzyme that is commonly seen in human erythrocytes. It helps our respiratory system remove CO2 by increasing CO2 solubility in the bloodstream. CA catalyzes the gas form of CO2 to ionic form of bicarbonate as dissolved inorganic compound.

We used CA to improve the dissolution rate of CO2 in the algal culture medium.

Dissolved bicarbonate (HCO3-) is transported into the algal cell along with gaseous CO2. And HCO3- is accumulated and concentrated in the cells until conversion back to CO2, which is used by Rubisco to fix carbon into glucose. This pathway increases CO2 concentration and prevents the deleterious oxygenation effect in carboxysome.

In this project, we utilized human CA to facilitate CO2 transportation as DIC form in the culture media. Algae can take them up and then fix CO2 into biomass.

Cytochrome P450 2E1 (CYP2E1)

CYP2E1 is a member of cytochrome P450(CYP), which plays a role in metabolizing the toxin and drug including alcohol, benzene, chloroform, 4-nitrophenol, acetone, etc. in the animal body. We use CYP2E1 in our system to break down VOCs (i.g., benzene and chloroform), and algae are capable of taking up the degraded small molecules, resulting in improving the indoor air quality by cleaning up VOCs.

Phenol is a product from benzene oxidation which is catalyzed by CYP2E1.

CO2 is one of the major metabolites generated in the process of chloroform degradation. CYP2E1 plays a vital role in the first step followed by spontaneous reactions to make CO2.

Phenol and CO2 can be naturally absorbed by algae and turned into biomass. We will use CYP2E2 as a biocatalyst to remove benzene and chloroform.

BIOBRICK CONSTRUCTION

Gene design & cloning

To produce CA and CYP2E1, we constructed biobricks consisting of PliaI promoter with a RBS and a reporter (GFP) or genes of interest (GOI) (i.g., CA, CYP2E1) followed by a terminator. The GOIs were tagged with 6xHis for further protein purification if needed.

PliaI(BBa_K823001) created by LMU-Munich in iGEM 2012 is a promoter of the liaHI operon on the genome of Bacillus subtilis, and it is activated by bacitracin as an inducer. We synthesized the gene of human carbonic anhydrase II (CA) or rabbit cytochrome P450 2E1 (CYP2E1) by Twist Bioscience. Then, DNA fragments were amplified by PCR and assembled with a terminator, followed by inserting to PliaI-RBS/pSB1C3.

Below are some of the gel data in the process of cloning CA and CYP2E1. All of the constructs have been further confirmed by DNA sequencing with primers of VF2 and VR.

GENE EXPRESSION & PURIFICATION

PliaI promoter induced by bacitracin

To know the activity of PliaI promoter in Bacillus subtilis, we made a construction (BBa_K2932004) by assembling PliaI with RBS-GFP(BBa_I13500), and then transfer the cassette of PliaI-RBS-GFP-Terminator to pBS0E, which is a replicative plasmid in Bacillus subtilis. GFP expression in Bacillus was tested by our induction procedure (see Notebook).

Our data showed that PliaI is a strong promoter with a low basal level, which is consistent with the result observed team LMU-Munich in 2012. After induction with bacitracin, GFP intensity is expressed 2.81-fold higher than uninduced cells. The pellets can be easily seen with naked eyes. (Left to right: PliaI, PliaI + Bacitracin, PliaI-RBS-GFP, PliaI-RBS-GFP + Bacitracin)

CA & CYP2E1 gene expression

To express genes of CA and CYP2E1 in Bacillus subtilis, we transferred the DNA fragments of PliaI-RBS-CA-Tr and PliaI-RBS-CYP2E1-Tr to pBS0E vector, and transformed Bacillus subtilis 168 with the resulting plasmids.

Total lysates of Bacillus expressing CA and CYP2E1 were subjected to SDS-PAGE and stained with Coomassie blue. The results showed below (CA data is our work shown in the left, and CYP2E1 data is a collaboration work with NCTU-Formosa shown in the right). CA protein was observed after induction around 30kDa as the same size of predicted molecular weight. CYP2E1 protein has a molecular weight of 55kDa and showed as a band overlapped with a lot of proteins located between 48-63 kDa. Further confirmation is needed to make sure the expression of CYP2E1 and optimal induction procedure.

CA protein purification

After discussion with iGEM team CSMU_Taiwan, they recommended purifying proteins prior to conducting functional assay. We prepared the induced CA lysates. They performed His-tag affinity-based Ni-NTA column chromatography to purify the protein.

Experiment procedure

↓ Equilibrate Ni-NTA resin in the column with 20mM Tris-HCH, 200mM NaCl, pH7.5
↓ Load protein lysates onto the column. The flow-through was collected.
↓ Wash the column with 20mM Tris-HC, 200mM NaCl, 5mM imidazole, pH7.5 The wash-through was collected.
↓ Elute the column with 20mM Tris-HC, 200mM NaCl, 20mM imidazole, pH7.5. The Elution #1 was collected.
↓ Elute the column with 20mM Tris-HC, 200mM NaCl, 200mM imidazole, pH7.5. The Elution #2 was collected.
↓ Elute the column with 20mM Tris-HC, 200mM NaCl, 500mM imidazole, pH7.5 The Elution #3 was collected.


The total lysates, flow-through, wash-through, Elution #1, #2, #3 were subjected to SDS-PAGE and Coomassie Blue staining as well as Western Blotting with the anti-His antibody, which of all were conducted by CSMU_Taiwan. The data presented below gave a clear evidence of the CA protein induction, expression and purification.

CA FUNCTIONAL ASSAY

CA function test

To test the function of CA, we added the purified CA into ddH2O and pump the ambient CO2 (~450 ppm) into the water. The pH level and CO2 dissolution rate were measured by pH meter and CO2 sensor, respectively. Based on the catalyzation reaction of CA, if CA works, CO2 level will drop and pH value of water should decrease.

The data was presented below. The left figure showed the CO2 fastly dissolved into water and CO2 elevated to the normal level after 8 min due to CO2 saturation in the water. The right figure indicated the pH value of water supplemented with CA decreased quicker than the one without CA, confirming the function of CA.

Microalgae purification system demonstration

Our design concept is based on the microalgae purification photobioreactor system. To enhance CO2 removal and algal biomass generation, the bioactive lysates of Bacillus which was genetically engineered to produce CA are supplemented to the system. The system is able to refresh air by removing CO2 efficiently, which is fixed by algae to generate biomass.

We compared the cost of production and application between the commercial / home-made purified protein and our unpurified bacterial lysates. See the comparison table below. The cost could be reduced dramatically up to near 10 fold in the application of our two systems of CAir™ and CAir Pro™ (see Hardware)

Analysis device setup

To determine the efficiency of our system, the CO2 & biomass analysis device was set up. We used parameters of CO2 concentrations, air flow rate, algae culture volume, light intensity and test periods as indicated in the schematic diagram.

The CO2 consumption and the biomass generation are two indexes for the efficiency of our system and should be improved in the presence of active CA.

Among microalgae, cyanobacteria could perform photosynthesis with light in 24 hours a day. Unicellular eukaryotic algae like Chlorella spp. need light:dark cycles to adjust the photosynthesis system (comments by an algae expert, Ms. HUANG, YA-EN). Therefore, we chose cyanobacteria for our targets. We tested two cyanobacterial strains. One is lab strain, Synechococcus elongatus PCC7942, obtained from National Chiao Tung University. The other is an edible strain, Spirulina spp., purchased as food supplements from pharmacy store. We ground the pill made of Spirulina into powder and put it into BG-11 media in the same condition of culturing the lab strain.

Experiment #1

Strain: Synechococcus elongatus PCC7942
Index: CO2 consumption
Enzyme: 1ml of Bacillus raw lysates containing CA (CA was estimated at 50mg)
Starting OD730 of algae: 0.5
Culture volume: 100 ml or 300ml of BG-11
Input of CO2: 1000 ppm or 2000 ppm
Air flow rate: 300 ml/min or 600 ml/min
Light: 5000 lux
Time period: 1hr

Result:
When providing 1000 ppm of CO2, 100 ml of algae consumed 37.2% or 59.6% CO2 wither without CA, respectively. The CA enhances 60.1% efficiency of CO2 removal. However, 300 ml of algae absorbed more CO2 (84.4% without CA, 90.3% with CA). When increasing CO2 input to 2000 ppm, 300 ml of algae with CA improved 33.3% of CO2 consumption compared to the group without CA. If increasing more CO2 input by tuning up air flow rate from 300 ml/min to 600 ml/min, a significant improvement (78.3%) was obtained by algae culture in the presence of CA.

Experiment #2

Strain: Synechococcus elongatus PCC7942
Index: Biomass generation
Enzyme: 1ml of Bacillus raw lysates containing CA (CA was estimated at 50mg)
Starting OD730 of algae: ~0.5
Culture volume: 300ml of BG-11
Input of CO2: ambient CO2 (~450ppm)
Air flow rate: 300 ml/min or 600 ml/min
Light: 3000 lux
Time period: 24hr

Result:
The table below presented the OD730 values of Synechococcus growing in the absence or presence of CA.

According to the equation of the conversion of OD730 value to Biomass (g/L/day), we obtained the data that the generation of biomass in the culture with CA increased 3.24x more than the culture without CA. The result further confirmed that the increased CO2 consumption which is facilitated by CA is converted to biomass by algal CO2 fixation.

Experiment #3

Strain: Spirulina spp.
Index: CO2 consumption
Enzyme: 1ml of Bacillus raw lysates containing CA (CA was estimated at 50mg)
Starting OD730 of algae:~0.5
Culture volume: 300ml of BG-11
Input of CO2: 850 ppm
Air flow rate: 600 ml/min
Light: 5000 lux
Time period: 30 min or 1hr

Result:
Our microalgae system was tested with an edible strain, Spirulina. In consistent with the result in the data of Synechococcus, the Spirulina with CA consumed CO2 more efficiently than ones with WT bacterial lysates. And the improvement because of CA can be maintained at least for 1 hr. The result shown below is one of the representative data.

Summary

Our goal is to create a microalgae purification system, in which algae efficiently consuming CO2 and growing biomass could be applied to general products such as food supplements and fertilizers, etc. So we are producing protein catalysts instead of genetically engineering algae (GMO). In our experiments, we successfully demonstrated the enzyme function of the purified CA and Bacillus lysates containing CA. In addition, the CO2 consumption was improved significantly in the microalgae purification system with Synechococcus and Spirulina, suggesting that this system is working and has a potential for many applications.

CYP2E1 FUNCTIONAL ASSAY

Method of product measurement

CYP2E1 catalyzes benzene conversion to form phenol, which could be taken up by algae. To measure the function of CYP2E1, we used Emerson reagent to measure the phenol production.

4-aminoantipyrine colorimetric reaction (Emerson reaction)

Emerson reaction is describing 4-aminoantipyrine (4-AAP) oxidation with phenol in alkaline condition catalyzed by oxidative potassium ferricyanide (K3[Fe(CN)6]) to produce p-quinoneimide adduct in red color.

To test Emerson reaction, we prepare phenol solution (83g/L, equals to 0.88M, i.e., phenol solubility in water) with 10X serial dilution. The red color product of the reaction can be measured at OD580. The color changed from yellow, orange to red color depends on the concentration of phenol.

The calibration curve presented in the figure below is made in the scatter plot. The concentration of phenol (mg/L) (Y) can be calculated from the values of OD580 by the equation of Y = 0.00003*e^(16.202*X)

CYP2E1 function test

Benzene is one of substrates of CYP2E1. Because of the toxicity of benzene, a collegiate iGEM team, Tunghai-TAPG helped us conduct the analysis in a specialized laboratory in Department of Chemistry in the university.

Experiment procedure

↓ prepare CYP2E1 and WT lysates
↓ prepare 1.79 g/L of benzene (i.e., benzene solubility in water)
↓ add 90μl of benzene solution with 10X serial dilution to each well
↓ incubate with 40μl of CYP2E1 or WT Bacillus lysates at room temperature for 30 min
↓ transfer 90μl of the mixture to a new well
↓ add 90μl of solution I (1% of 4-aminoantipyrine in KOH solution, pH=9~10
↓ then add 45μl of solution II (4% of K3[Fe(CN)6])
↓ measure at OD580

The data showed that the values measured at OD580 are higher in the group of benzene with CYP2E1 compared to controls of benzene without CYP2E1, indicating that the phenol is converted from benzene by CYP2E1. The benzene has a basal effect in Emerson reaction. The values of OD580 of Benzene are regarded as background and are subtracted for study the phenol generation.

Next, according to the calibration curve of phenol analysis in Emerson reaction. We converted the OD values of phenol formation to benzene degradation. The result suggested that CYP2E1 in the bacterial lysate can convert 0.09 - 0.47 μg/L of benzene to phenol, implying the possibility of CYP2E1 application in benzene removal in our microalgae purification system.

Microalgae purification system demonstration

Finally, we’d like to know the phenol consumption by algae in our system. We incubated algae with a serial 10x dilution of phenol solution as did in the analysis of the calibration curve, followed by culturing algae with light at 37°C for 1hr. Then, the algae culture media was centrifuged to discard the algal cells, and the supernatants was subjected to Emerson reaction assay. As data shown, the OD values dropped significantly in the group of phenol with algae in a dose-dependent manner, indicating that phenol is consumed by algae.

Using the calibration curve of phenol concentration to OD values, we obtained the data that algae can take up 0.15 - 0.34 μg/L of phenol in culture media. The result is consistent with the study by M. Wurster, et al. in 2003, proving the algae is capable of removing phenol in the environment.

Summary

We successfully demonstrated the CYP2E1 can oxidize benzene to phenol, which can be absorbed by algae. These findings extend the possibility of applying the enzymes to microalgae purification system to remove various pollutants or toxic chemicals, which are naturally unable to be absorbed by algae.

DISCUSSION

Carbonic anhydrase (CA) application in CO2 capture and sequestration was studied intensively. Joel K. J. Yong, et al. studied in 2015 about CO2 capture in the chemical absorption process using CA. The CA is immobilized within the gas absorber and enhance CO2 capture performance. A review paper by Madhumanti Mondal, et al. in 2016 summarizes using CA in biological carbon capture through microalgae. In our study, we applied this technique to our system and engineered Bacillus to produce CA based on synthetic biology. And we make an effort to bring the product and application to the real world.

Cytochrome P450 2E1 (CYP2E1) used in benzene and chloroform removal is first studied by Sharon L. Doty, et al. in 2007 and published on the distinguished PNAS journal. The authors demonstrated that CYP2E1-transgenic tree, Populus alba, has the ability to remove benzene at 0.28 μg/h. Based on our study in microalgae purification system, it is for the first time to demonstrate algae is able to take up phenol converted from benzene in the process of CYP2E1 catalyzation. The algae can indirectly absorb benzene at 0.95 μg/h, which improve the efficiency up to 4.7 fold. Our result showed that the microalgae system has great potential of removing unwanted pollutants efficiently with extracellular enzymes, which convert the pollutants to substrates absorbed by algae.

Microalgae purification system is getting increasingly attention. Our work demonstrated that the system can be improved by using bioactive enzymes (CA and CYP2E1) to enhance CO2 consumption rate and acquire the ability to remove various pollutants. Our system is multifunctional using enzymes, low-cost using Bacillus lysates and applicable using edible algae rather than GMOs.

Reference

  1. Cornelia Geers, and Gerolf Gros “Carbon Dioxide Transport and Carbonic Anhydrase in Blood and Muscle“ Physiological Reviews - American Journal of Physiology. 2000; 80(2):681-715. doi: 10.1152/physrev.2000.80.2.681
  2. J. V. Moroney and A. Somanchi “How Do Algae Concentrate CO2 to Increase the Efficiency of Photosynthetic Carbon Fixation?” Plant Physiol. 1999 Jan; 119(1): 9–16. doi:10.1104/pp.119.1.9
  3. Frank J. Gonzalez. “CYP2E1” Drug Metabolism and Disposition 2007, 35 (1) 1-8; doi:10.1124/dmd.106.012492
  4. M. Wurster, S. Mundt, E. Hammer, F. Schauer, U. Lindequist “Extracellular degradation of phenol by the cyanobacterium Synechococcus PCC 7002” Journal of Applied Phycology. 2003; 15(2–3) pp 171–176 doi:10.1023/A:1023840503605
  5. Philipp F. Popp, Mona Dotzler, Jara Radeck, Julia Bartels & Thorsten Mascher. "The Bacillus BioBrick Box 2.0: expanding the genetic toolbox for the standardized work with Bacillus subtilis" Scientific Reports 2017; 7(1):15058 doi: 10.1038/s41598-017-15107-z.
  6. Ashley E. Beck, Kristopher A. Hunt and Ross P. Carlson "Measuring Cellular Biomass Composition for Computational Biology Applications" Processes 2018, 6(5), 38; doi:10.3390/pr6050038
  7. Selvakumar, Paulraj Mosae. “Phenol Sensing Studies by 4-Aminoantipyrine Method-A Review.” Organic & Medicinal Chemistry International Journal 2018:5(2) doi:10.19080/omcij.2018.05.555657.
  8. Joel K. J. Yong, Geoff W. Stevens, Frank Caruso, Sandra E. Kentish "The use of carbonic anhydrase to accelerate carbon dioxide capture processes" Journal of Chemical Technology & Biotechnology 2015:90(1) doi:10.1002/jctb.4502
  9. Madhumanti Mondal, Saumyakanti Khanra, O.N. Tiwari, K. Gayen G.N. Halder. "Role of carbonic anhydrase on the way to biological carbon capture through microalgae—A mini review" American Institute of Chemical Engineers Environ Prog, 2016: 35:1605–1615, doi:10.1002/ep.12394
  10. Sharon L. Doty, C. Andrew James, Allison L. Moore, Azra Vajzovic, Glenda L. Singleton, Caiping Ma, Zareen Khan, Gang Xin, Jun Won Kang, Jin Young Park, Richard Meilan, Steven H. Strauss, Jasmine Wilkerson, Federico Farin and Stuart E. Strand. "Enhanced phytoremediation of volatile environmental pollutants with transgenic trees." Proc Natl Acad Sci USA. 2007;104(43):16816-21. doi: 10.1073/pnas.0703276104