Part:BBa_I746916:Experience

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

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.

User Reviews

UNIQ984d47339f13944b-partinfo-00000004-QINU UNIQ984d47339f13944b-partinfo-00000005-QINU