Team:Linkoping Sweden/Contribution
Characterization
Characterization
During this project the team characterized three different biobricks: pCons-eforRed (BBa_K864402), sfGFP (BBa_I746916) and AsPink (BBa_K1033933).pCons-eforRed
This 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.
Figure 1. The picture to the left depicts a cell culture of BL21 (DE3) pCons-eforRed (left tube) versus a negative control (right tube) with BL21 (DE3). The top right picture displays a pellet of BL21 (DE3) with pCons-eforRed in UV-light. The right picture is a pellet of BL21 (DE3) 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).
Figure 2. Colonies in the same host as previously is presented in white light (right side) and in 302 nm UV-light (left side).
Oxygen dependency
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.
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.
Figure 3. To the left is a spectrometry experiment of BL21´s (DE3) protein production of eforRed with varying access to oxygen. The y-axis depicts the relative fluorescence intensity and the x-axis represents the time up to 24 hours. The numbers represent the amount of holes pierced in the protective plastic film. The plate with eforRed can be seen on the right pictures left side and the one on the right side is E.coli BL21 (DE3) with a different expression system used to test a strong green/yellow fluorescent protein. Both plates were illuminated in 302 nm UV-light.
Molecular weight
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.
sfGFP
In this characterization, the absorbance and fluorescence was demonstrated in different chassis. The protein expression was also measured in different bacteria. An important thing to note is that the sfGFP is fused to the CBDcipA (BBa_K3182200). 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) 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.
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.
Figure 5. A culture with induced BL21 (DE3) expressing CBD-sfGFP on an UV-table emitting at 302 nm.
Compatibility in Vibrio natriegens
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.
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.
Figure 6. Picture to the right depicts a LB-agar dish with V.natriegens (Vmax) expressing pUC19 CBD-sfGFP. To the left is a control with Vmax 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 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.
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.
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.
AsPink
To visualize the absorbance of AsPink (BBa_K1033933) on a petri dish, BL21 (DE3) colonies containing the pUC19-pCons-AsPink part (BBa_K3182100), which was used in the pink-white screening, were incubated for 16 hours in 37 °C. The pCons-AsPink construct was removed and (Magainin 2) and BBa_K3182104 (CHAP) was inserted into the vector. The pink colonies contain AsPink and indicate a religated pCons-AsPink. The white colonies indicate a successful ligation. Below (Figure 8) is a picture of the pink-white screening.
Figure 8.
BL21 (DE3) bacteria used for pink-white screening. The white colonies indicate a successful ligation and the pink colonies is AsPink expressing bacteria which signals a false-positive ligation.
