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).

CA FUNCTIONAL ASSAY

CYP2E1 FUNCTIONAL ASSAY

DISCUSSION