Overview

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

1. 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
2. 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.
3. 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