CharacterizationDuring this project the team characterized three different biobricks: pCons-eforRed (BBa_K864402), sfGFP (BBa_I746916) and AsPink (BBa_K1033933).
pCons-eforRedThis part was verified by displaying its absorbance, fluorescence, oxygen dependency and molecular weight.
Fluorescence and absorbance
To verify eforRed's (BBa_K864402) absorbance and emission, the construct was heat shocked into Escherichia coli, using the strain BL21 (DE3), and grown in a Falcon tube O.N. in 37 °C at 25 µg/mL chloramphenicol. Cotton plugs was used as corks for the Falcon tube. The bacterial solution was compared to a negative control with only BL21 (DE3)(Figure 1, left side) which showed the red color of eforRed in a bacterial solution. Thereafter, the eforRed expressing bacteria was centrifuged at 12 000 g for 10 minutes which displayed a burgundy colour (Figure 1, top-right corner). The pellet was also placed on an UV-table emitting light at 302 nm (Figure 1, down-right corner) and exhibited a pink glowing colour. These experiments verifies eforRed fluorescent effect as well as its absorbance in white light.
Further characterization was performed in order to demonstrate the absorbance and fluorescence of eforRed. BL21 (DE3) containing pCons-eforRed were spread on a petri dish containing 25 µg/ml chloramphenicol and was photographed in white light and on an UV-table emitting 302 nm (Figure 2). The results were the same as above, in white light (Figure 2, right) the cultures had a burgundy color and on the UV-table the eforRed expressing bacteria exhibited a pink glowing colour (Figure 2, left).
To test the oxygen dependency of the protein production of eforRed in BL21 (DE3), the bacteria containing pCons-eforRed was grown O.N. to 2 OD600 and diluted to 0.49 OD600 with LB-miller. The bacteria was placed in a 96-well plate in replicates of 4 with 200 µL in each well. The oxygen access was varied by piercing different numbers of holes (0, 1, 2, 3 and 4) in the plastic film of the 96-well plate. A spectrometry experiment was conducted measuring the fluorescence (excitation 589, emission 609) in 37 °C for 24 hours and the experiment (Figure 3) showed that the access to oxygen effects the folding of eforRed and that 4 holes gave the highest yield.
A verification of eforRed was performed by SDS-PAGE. pCons-eforRed expressed in E.coli BL21 (DE3) which were later sonicated (30 sec on/30 sec off in 30% amplitude for 4 minutes). Thereafter, an SDS-PAGE was performed on the lysate (Figure 4) with a BioRad Precision Plus Protein Dual Color Standards protein ladder. The electrophoresis (Figure 4) verified the existance of eforRed in the lysate with a visible band between 25 and 37 kD which corresponds to the molecular weight of eforRed (26.1 kDa).
Figure 4. A 4-20% SDS-PAGE of sonicated BL21 (DE3) lysate with pCons-eforRed. BioRad´s Precision Plus Protein Dual Color Standards was used as the protein ladder.
Fluorescence in BL21 (DE3)
To verify the fluorescence of sfGFP (BBa_I746916) fused to CBDcipA(BBa_K3182200), BL21 (DE3) containing CBD-sfGFP was grown in 1 liter LB-miller with 25 µg/ml chloramphenicol. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was used to induce the culture at a final concentration of 1 mM and the culture was incubated O.N. in 37 °C after the induction. Thereafter, the CBD-sfGFP expressing bacteria was placed on an UV-table emitting light at 302 nm (Figure 5). The figure shows CBD-sfGFP´s fluorescence.
In order to see if sfGFP worked in Vibrio natriegens using the strain Vmax, CBD-sfGFP (BBa_K3182108) and CBD-pCons-Aspink (BBa_K3182100) was ligated into the pUC19 vector and heat shocked into Vmax.Thereafter, the bacteria was spread onto LB-miller V2 agar dishes with 200 µg/ml carbenicillin and incubated in 37 °C for 16 hours. Both plates was put on an UV-table and illuminated at 302 nm (Figure 6). The figure below shows that the CBD-sfGFP bacteria, in comparison to the control CBD-pCons-AsPink, displays a strong green fluorescent color which verified that pUC19-CBD-sfGFP could successfully be heat shocked and expressed in Vmax.
To measure the protein expression of pT7-CBD-sfGFP in different bacteria and carbenicillin concentrations. BL21 (DE3) and Vibrio natriegens , using the strain Vmax, was grown in Falcon tubes to 0.5 OD600. Vmax was grown with two different carbenicillin concentrations, 200 and 600 µg/mL, while BL21 (DE3) had the same carbenicillin concentration of 100 µg/mL carbenicillin. The bacteria was induced with 1 mM IPTG and placed in a 96-well plate in 4 replicates with 200 µL per well. A spectrometry experiment was conducted and measured the fluorescence (excitation 470 nm,emission 550 nm) during 16 hours in 37 °C. The results seen below (Figure 7) shows that expression in Vmax with 600 µg/mL carbenicillin gave the highest protein yield. The most probable explanation for the increased protein yield for Vmax at 600 µg/mL carbenicillin is partially caused by the higher protein production of Vmax compared to BL21 (DE3). Another important factor was the use of an optimal concentration of carbenicillin (600 µg/mL) for Vmax which retained the plasmid more efficiently than Vmax at 200 µg/mL carbenicillin.