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Applications of BBa_I746916

2019 iGEM team Linkoping Sweden

2019 iGEM team Linkoping Sweden validated this part.
Summary: In this contribution we verified the fluorescence of CBD-sfGFP, studied the compatibility of CBD-sfGFP in Vibrio natriegens and measured the expression of CBD-sfGFP in different chassis. An important thing to note is that the sfGFP is fused to the CBD_cipA (BBa_I746916). However, as can be seen below, the sfGFP still maintained a high fluorescence and was able to be folded correctly.

Fluorescence in BL21 (DE3)
To verify the fluorescence of sfGFP (BBa_I746916), 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 302 nm (Figure 5). The picture shows CBD-sfGFP´s strong fluorescence at 302 nm UV-light.

Figure 5. 1 liter LB-miller with induced BL21 (DE3) expressing CBD-sfGFP on an UV-table emitting UV-light at 302 nm.

Compatibility in Vibrio natriegens
In order to see if sfGFP worked in Vibrio natriegens using the strain V_max, CBD-sfGFP (BBa_K3182108) and CBD-pCons-Aspink (BBa_K3182100) was ligated into the pUC19 vector and heat shocked into V_max.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 in 302 nm (Figure 6). The picture 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 V_max.

Figure 6. Picture to the right depicts a LB-agar dish withV_maz expressing pUC19 CBD-sfGFP. To the left is a control with V_max expressing PUC19 CBD-pCons-Aspink. Both dishes was placed on an UV-table and illuminated in 302 nm.

Protein expression in different chassis
To measure the protein expression of T7-CBD-sfGFP in different bacteria and carbenicillin concentrations. BL21 (DE3) and Vibrio natriegens , using the strain V_max, was grown in Falcon tubes to 0.5 OD₆₀₀. V_max 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 V_max with 600 µg/mL carbenicillin gave the highest protein yield. The most probable explanation for the increased protein yield for V_max at 600 µg/mL carbenicillin is partially caused by the higher protein production of V_max compared to BL21 (DE3). Another important factor was the use of an optimal concentration of carbenicillin (600 µg/mL) for V_maxwhich retained the plasmid more efficiantly than V_max at 200 µg/mL carbenicillin.

Figure 7. CBD-sfGFP expression in different chassis. The orange line represent Vmax at 600 µg/mL carbenicillin, blue represents Vmax at 200 µg/mL carbenicillin, and green represents E. coli BL21 at 100 µg/mL carbenicillin. The y-axis depicts RFU and the x-axis represents the time over 28 hours.

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