Team:Mingdao/Demonstrate

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JUDGING FORM ⇗

Biobrick Function

Biobrick Construction

Gene Expression & Purification

CA

CYP2E1

Discussion

To increase the efficiency of our algae system to reduce indoor pollutants like 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 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 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 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 in addition to 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 utilize human CA to facilitate CO2 transportation as DIC form in the culture media. Algae can take them up followed by fixing 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 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 (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-GFP, PliaI-GFP+Bacitracin)

CA & CYP2E1 gene expression

To express genes of CA and CYP2E1 in Bacillus subtilis, we transfer 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 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

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.

CYP2E1 FUNCTIONAL ASSAY

DISCUSSION