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===Applications of BBa_I746916===
 
===Applications of BBa_I746916===
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<h2> 2019 iGEM team Linkoping Sweden </h2>
 
<h2> 2019 iGEM team Linkoping Sweden </h2>
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2019 iGEM team Linkoping Sweden validated this part.<br>
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Summary: In this contribution we verified the fluorescence of CBD-sfGFP, studied the compatibility of CBD-sfGFP in <i> Vibrio natriegens </i> and measured the expression of CBD-sfGFP in different chassis. An important thing to note is that the sfGFP is fused to the CBD<sub>cipA</sub> <html>(<a href="https://parts.igem.org/Part:BBa_K3182200">BBa_I746916</a>)</hmtl>. However, as can be seen below, the sfGFP still maintained a high fluorescence and was able to be folded correctly.<br><br>
  
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[[Image:T--Linkoping_Sweden--CBD-sfGFPstorodl4.jpeg|300px|thumb|left|<b><i>Figure 1.</i></b> 1 liter growth medium containing CBD-sfGFP BL21 (DE3) (induced?) on a UV-table. ]]  
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<b style=font-size:120%;> Fluorescence in BL21 (DE3)</b> <br>
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<br> Pictures above shows induced BL21 DE3 and Vibrio natriegens Vmax expressing CBD-sfGFP. The picture to the left is a 1 liter growth medium with E.coli BL21 expressing CBD-sfGFP. The picture in the middle is a depiction of an induction gradient with BL21 with varying IPTG concentrations between 0.05-1.0 mM. The picture to the left is a 1 liter culture of induced Vibtio natriegens
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To verify the fluorescence of sfGFP (<a href="https://parts.igem.org/Part:BBa_I746916">BBa_I746916</a>), 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.
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</html>[[Image:T--Linkoping_Sweden--CBD-sfGFPstorodl4.jpeg|300px|thumb|left|<b>Figure 5.</b> 1 liter LB-miller with induced BL21 (DE3) expressing CBD-sfGFP on an UV-table emitting UV-light at 302 nm.]] <html>
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<b style=font-size:120%;> Compatibility in <i> Vibrio natriegens </i><br> </b>
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In order to see if sfGFP worked in <i>Vibrio natriegens</i> using the strain V<sub>max</sub>, CBD-sfGFP (<a href="https://parts.igem.org/Part:BBa_K3182108">BBa_K3182108</a>) and CBD-pCons-Aspink (<a href="https://parts.igem.org/Part:BBa_K3182100">BBa_K3182100</a>) was ligated into the pUC19 vector and heat shocked into  V<sub>max</sub>.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<sub>max</sub>.
  
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The chosen IPTG concentrations were XX mM for e.coli and Vibrio natriegens
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</html>[[Image:T--Linkoping_Sweden--CBD-sfGFPvibrio.jpeg|600px|thumb|left|<b>Figure 6.</b> Picture to the right depicts a LB-agar dish with<i>V<sub>maz</sub></i> expressing pUC19 CBD-sfGFP. To the left is a control with V<sub>max</sub> expressing PUC19 CBD-pCons-Aspink. Both dishes was placed on an UV-table and illuminated in 302 nm.]]<html>
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<b style=font-size:120%;> Protein expression in different chassis</b><br>
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To measure the protein expression of T7-CBD-sfGFP in different bacteria and carbenicillin concentrations. BL21 (DE3)</i> and <i> Vibrio natriegens </i>, using the strain V<sub>max</sub>, was grown in Falcon tubes to 0.5 OD<sub>600</sub>. V<sub>max</sub> 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<sub>max</sub> with 600 µg/mL carbenicillin gave the highest protein yield. The most probable explanation for the increased protein yield for V<sub>max</sub> at 600 µg/mL carbenicillin  is partially caused by the higher protein production of  V<sub>max</sub> compared to BL21 (DE3). Another important factor was the use of an optimal concentration of carbenicillin (600 µg/mL) for V<sub>max</sub>which retained the plasmid more efficiantly than V<sub>max</sub> at 200 µg/mL carbenicillin.  </div>
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</html>[[Image:T--Linkoping_Sweden--CBD-VmaxEcoliMeasurement.png|600px|thumb|left|<b>Figure 7.</b> 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.]]<html>
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  The induction gradient for Vibrio is more correlated with the IPTG concentration then E.Coli suggesting that the induction windows are different between the bacteria with CBD-sfGFP.
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Latest revision as of 17:03, 20 October 2019


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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 CBDcipA (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 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 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 Vmax.
Figure 6. Picture to the right depicts a LB-agar dish withVmaz 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 T7-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 Vmaxwhich retained the plasmid more efficiantly 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.



























































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