Indoor air pollutants are a serious problem, and we created a microalgae purification photobioreactor device, named CAir^TM , which is one of the best solutions to it. To produce enzymes that can be applied to CAir^TM system, we developed a CAir^TM kit. The 1st generation of the kit contains materials and instruction for removing CO2 and VOCs.
The two main features in the kit are CA and CYP2E1. CA is an enzyme enhancing CO2 dissolution rate for algae uptake. CYP2E1 is an enzyme that can detoxify benzene and chloroform.
To make materials in the CAir^TM kit easy to use, we designed a GFP reporter under PliaI promoter. The protein induction condition and expression level can be tested with this plasmid.
Buy a CAir^TM (see Prototype & Entrepreneurship) with a CAir^TM kit, everyone is able to refresh the air by themselves.

Part collection

The CAir^TM kit provides the transformed Bacillus subtilis carrying the DNA materials on the vector of pBS0E, a replicative plasmid in Bacillus subtilis. The table lists the basic parts and composite parts on the vector of pSB1C3, which are created during the work in iGEM 2019. The functions of the parts are tested on the backbone of pBS0E.

Protein expression test

Part name: PliaI + RBS + GFP + terminator
Part cat. no.: BBa_K2932004

To know the activity of PliaI promoter in Bacillus subtilis, we made a construct (BBa_K2932004) by assembling PliaI with GFP(BBa_E0040), 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.

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-GFP, PliaI-GFP+Bacitracin)

User instruction

GFP protein induction procedure

↓ culture Bacillus subtilis 168 carrying the plasmid of PliaI + RBS + GFP + terminator/pBS0E in LB supplemented with erythromycin (1 μg/ml) and lincomycin (25 μg/ml) O/N at 37°C, shaking at 170 rpm
↓ transfer 3 ml to 50 ml LB with antibiotics in 250 ml flask
↓ measure OD650
↓ shake at 200rpm,37°C until OD650 between 0.5~0.7
↓ add 30μg/ml of bacitracin for induction at 25°C, shaking at 100 rpm in the incubator for 18.5 hr

CA protein induction procedure

↓ culture Bacillus Subtilis 168 carrying the plasmid of PliaI + RBS + CA + terminator/pBS0E in LB + antibiotics O/N at 37°C, shaking at 170 rpm, supplied with 60μM ZnSO4
↓ transfer 3 ml to 50 ml LB+Antibiotics in 250 ml flask with 60μM ZnSO4
↓ measure OD650
↓ shake at 200rpm,37°C until OD650 between 0.5~0.7
↓ add 400μM ZnSO4 and 50μl of 30μg/ml Bacitracin for induction at 25°C, shaking at 100 rpm for 18.5 hr

CYP2E1 protein induction procedure

↓ culture Bacillus subtilis 168 carrying the plasmid of PliaI + RBS + CYP2E1 + terminator/pBS0E in LB + Amp (100 μg/ml)O/N at 37°C, shaking at 170 rpm, supplemented with thiamine 1mM and 75mg/L of 5-aminolevulinic acid
↓ transfer 3 ml to 50 ml LB+Antibiotics with all the supplements in 250 ml flask
↓ measure OD650
↓ shake at 200rpm,37°C until OD650 between 0.5~0.7
↓ add 50 μl of 30ug/ml Bacitracin for induction at 25°C, shaking at 100 rpm for 18.5 hr

Bacterial lysate preparation

↓ culture Bacillus subtilis 168 carrying the plasmid of PliaI + RBS + CA + terminator/pBS0E or PliaI + RBS + CYP2E1 + terminator/pBS0E in LB + AMP (100 μg/ml) O/N at 37°C
↓ perform the protein induction procedure
↓ centrifuge at 15000 xg
↓ discard the supernatant
↓ add 3ml of PBS buffer
↓ add 1.5ml of 0.1-mm disruption beads, 10μl of DNAse I and 30μl of PMSF
↓ vortex for 1 min, then put on ice for 30 sec, repeat 8 times.
↓ centrifuge at 15000 xg for 2 min

References

Banerjee AL1, Swanson M, Mallik S, Srivastava DK. “Purification of recombinant human carbonic anhydrase-II by metal affinity chromatography without incorporating histidine tags.” Protein Expr Purif. 2004 Oct;37(2):450-4. doi: 10.1016/j.pep.2004.06.031
Pritchard, Michael P., et al. “A General Strategy for the Expression of Recombinant Human Cytochrome P450s InEscherichia ColiUsing Bacterial Signal Peptides: Expression of CYP3A4, CYP2A6, and CYP2E1.” Archives of Biochemistry and Biophysics, vol. 345, no. 2, 1997, pp. 342–354., doi:10.1006/abbi.1997.0265.
Peter E. Vandeventer, et al. “Mechanical Disruption of Lysis-Resistant Bacterial Cells by Use of a Miniature, Low-Power, Disposable Device” J Clin Microbiol. 2011 Jul; 49(7): 2533–2539. doi: 10.1128/JCM.02171-10

Best Basic parts

Part name:CYP2E1, rabbit cytochrome P450 2E1 ( optimized for Bacillus preferred codon)

Part cat. no.:BBa_K2932000

Biological function

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

Benzene oxidation

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

Gene cloning and protein expression

We optimized the gene codon of rabbit cytochrome P450 2E1 (CYP2E1) based on Bacillus subtilis codon usage preference and synthesized the DNA fragment by Twist Bioscience. Then, DNA fragments were amplified by PCR and assembled with a terminator, followed by inserting to PliaI-RBS/pSB1C3.

Construct of PliaI-RBS-CYP2E1-Tr/pSB1C3

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

Protein induction procedure

↓ culture Bacillus subtilis 168 carrying the plasmid of PliaI + RBS + CYP2E1 + terminator/pBS0E in LB + Amp (100 μg/ml) O/N at 37°C, shaking at 170 rpm, supplemented with 1mM of thiamine and 75mg/L of 5-aminolevulinic acid
↓ transfer 3 ml to 50 ml LB+Antibiotics with all the supplements in 250 ml flask
↓ measure OD650
↓ shake at 200rpm,37°C until OD650 between 0.5~0.7
↓ add 50 μl of 30ug/ml Bacitracin for induction at 25°C, shaking at 100 rpm for 18.5 hr

After protein induction by bacitracin, the total lysates of Bacillus expressing CYP2E1 were subjected to SDS-PAGE and stained with Coomassie blue. CYP2E1 protein has a molecular weight of 55kDa and shown 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. (The work of SDS-PAGE was collaborated with team NCTU-Formosa)

Emerson reaction assay (4-aminoantipyrine colorimetric 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 functional assay

Benzene is one of the 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 the 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 plus 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 analyzing 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 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 were 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

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 to remove unwanted pollutants efficiently with extracellular enzymes, which convert the pollutants to substrates absorbed by algae.

References

Frank J. Gonzalez. “CYP2E1” Drug Metabolism and Disposition 2007, 35 (1) 1-8; doi:10.1124/dmd.106.012492
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
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.
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.
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
Sander, R. “Compilation of Henrys Law Constants (Version 4.0) for Water as Solvent.” Atmospheric Chemistry and Physics, vol. 15, no. 8, 2015, pp. 4399–4981., doi:10.5194/acp-15-4399-2015.

Best Composite Part

Part name:PliaI + RBS + CA + terminator

Part cat. no.:BBa_K2932003

Biological Function

Carbonic anhydrase (CA) is an enzyme which can be commonly seen in human erythrocytes, it helps the respiratory system to remove CO2 by increasing CO2 solubility in the bloodstream. The CA catalyzes the gas form of CO2 to the ionic form of bicarbonate as a dissolved inorganic compound.

Gene Cloning and Protein Expression

Human carbonic anhydrase II (BBa_K2932001) is well-studied and is one of the most efficient enzymes catalyzing the rapid conversion of CO2 to bicarbonate (HCO3-), and we used it to improve the dissolution rate of CO2 in the algal culture medium. We got the gene sequence from NCBI and synthesized the DNA fragment by Twist Bioscience. Then, DNA fragments were amplified by PCR and assembled with a terminator, followed by inserting to PliaI-RBS/pSB1C3. CA was tagged with 6xHis at C terminus for further protein purification if needed.

Construct of PliaI-RBS-CA-Tr/pSB1C3

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

Protein induction procedure

↓ culture Bacillus Subtilis 168 carrying the plasmid of PliaI + RBS + CA + terminator/pBS0E in LB + antibiotics O/N at 37°C, shaking at 170 rpm, supplied with 60μM of ZnSO4
↓ transfer 3 ml to 50 ml LB+Antibiotics in 250 ml flask with 60μM of ZnSO4
↓ measure OD650
↓ shake at 200rpm,37°C until OD650 between 0.5~0.7
↓ add 400μM of ZnSO4 and 50μl of 30μg/ml Bacitracin for induction at 25°C, shaking at 100 rpm for 18.5 hr
After protein induction by bacitracin, total lysates of Bacillus expressing were subjected to SDS-PAGE and stained with Coomassie blue. CA protein was observed after induction around 30kDa as the same size of predicted molecular weight.

CA protein purification

↓ 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 and Elution #1, #2, #3 were subjected to SDS-PAGE and Coomassie Blue staining as well as Western Blotting with the anti-His antibody. The data presented below gave a clear evidence of the CA protein induction, expression and purification. (The work of purification, SDS-PAGE and WB for CA protein was collaborated with team CSMU-Taiwan)

CA Functional Assay

To test CA function, 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

CO2 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.

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 the 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 without or with CA, respectively. The CA enhance 60.1% efficiency of CO2 removal. However, 300 ml of algae absorbed more CO2 (84.4% without CA, 90.3% with CA) but with ignored CA function. 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 which showed 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

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.

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.

References

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
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
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.
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
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
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

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Mingdao High School

iGEM

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Phone: +886-4-23341797
EMAIL: biolab@ms.mingdao.edu.tw
No.497, Sec. 1, Zhongshan Rd., Wuri District, Taichung City 414, Taiwan (R.O.C.)

Team:Mingdao/Parts

Overview

Part collection

Protein expression test

User instruction

GFP protein induction procedure

CA protein induction procedure

CYP2E1 protein induction procedure

Bacterial lysate preparation

References

Best Basic parts

Biological function

Benzene oxidation

Gene cloning and protein expression

Protein induction procedure

Emerson reaction assay (4-aminoantipyrine colorimetric reaction)

CYP2E1 functional assay

Experiment procedure

Microalgae purification system demonstration

Summary

Discussion

References

Best Composite Part

Biological Function

Gene Cloning and Protein Expression

Protein induction procedure

CA protein purification

CA Functional Assay

Microalgae Purification System Demonstration

CO2 analysis device setup

Experiment #1

Experiment #2

Experiment #3

Summary

Discussion

References

Quote

Links

Follow us on

Contact us