Difference between revisions of "Team:Manchester/Proof"

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  <center>
 
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   <img src="https://static.igem.org/mediawiki/2016/4/4e/T--Manchester--proofofconceptfigure1.png" alt="figure 1" />
 
   <img src="https://static.igem.org/mediawiki/2016/4/4e/T--Manchester--proofofconceptfigure1.png" alt="figure 1" />
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  <i>Figure 1: Schematic representation of the assembly of Plasmid 1.</i>
 
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<p style="font-size:17px;" class="lineheight160"> This plasmid was constructed to obtain a constant expression of the AlcR protein. As seen in the figure, the constitutive promoter and the alcR were initially present in two different plasmids. They were first digested with the appropriate restriction enzymes, ligated and then transformed into DH5α. The final plasmid construct has a Ampicilin/Carbenicilin resistance. Upon obtaining positive confirmation of the transformants, we proceeded to transform the ligated product into BL21, a protein expression strain.   
 
<p style="font-size:17px;" class="lineheight160"> This plasmid was constructed to obtain a constant expression of the AlcR protein. As seen in the figure, the constitutive promoter and the alcR were initially present in two different plasmids. They were first digested with the appropriate restriction enzymes, ligated and then transformed into DH5α. The final plasmid construct has a Ampicilin/Carbenicilin resistance. Upon obtaining positive confirmation of the transformants, we proceeded to transform the ligated product into BL21, a protein expression strain.   
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  <center>
 
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   <img src="https://static.igem.org/mediawiki/2016/a/a0/T--Manchester--proofofconceptfigure2.png" alt="figure 2" />
 
   <img src="https://static.igem.org/mediawiki/2016/a/a0/T--Manchester--proofofconceptfigure2.png" alt="figure 2" />
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  <i>Figure 2: SDS-PAGE gel showing protein expression results. As shown by boxes in the gel, the 27kDa rfp protein can be clearly seen. However, no proteins can be seen at the 98kDa region where the AlcR protein is expected to be present.</i>
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<p style="font-size:17px;" class="lineheight160"> After successfully obtaining transformants with the BL21 strain, we proceeded to overexpress the protein and attempted to isolate and purify the 98kDa AlcR protein by SDS-PAGE. Unfortunately, after several attempts, we failed to see any visible protein band on the SDS-PAGE. This could be due to many factors, one of them being that the amount of expressed protein is not enough to be visualised on a SDS-PAGE.
 
<p style="font-size:17px;" class="lineheight160"> After successfully obtaining transformants with the BL21 strain, we proceeded to overexpress the protein and attempted to isolate and purify the 98kDa AlcR protein by SDS-PAGE. Unfortunately, after several attempts, we failed to see any visible protein band on the SDS-PAGE. This could be due to many factors, one of them being that the amount of expressed protein is not enough to be visualised on a SDS-PAGE.
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   <img src="https://static.igem.org/mediawiki/2016/f/f7/T--Manchester--proofofconceptfigure3.png" alt="figure 1" />
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   <img src="https://static.igem.org/mediawiki/2016/f/f7/T--Manchester--proofofconceptfigure3.png" alt="figure3" />
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  <i>Figure 3: Schematic representation of the assembly of Plasmid 2.</i>
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     <p style="font-size:17px;" class="lineheight160"> This plasmid is crucial in proving that our mechanism works. It consists of the alcA promoter, which has the AlcR binding site, and chromoproteins to produce a visible colour change. Similar to Plasmid 1,  the constitutive promoter and the chromoproteins were initially present in two different plasmids. They were first digested with the appropriate restriction enzymes, ligated and then transformed into DH5α. The final plasmid construct has a Chloramphenicol resistance.
 
     <p style="font-size:17px;" class="lineheight160"> This plasmid is crucial in proving that our mechanism works. It consists of the alcA promoter, which has the AlcR binding site, and chromoproteins to produce a visible colour change. Similar to Plasmid 1,  the constitutive promoter and the chromoproteins were initially present in two different plasmids. They were first digested with the appropriate restriction enzymes, ligated and then transformed into DH5α. The final plasmid construct has a Chloramphenicol resistance.

Revision as of 13:50, 14 October 2016

Manchester iGEM 2016


Proof of Concept




After successfully characterizing our choice of constitutive promoters and chromoproteins, we proceeded to obtain a working model of our mechanism. We first built plasmid 1 and plasmid 2 (Figure 1: How it works?) using the iGEM 3A assembly method.

Plasmid 1


figure 1
Figure 1: Schematic representation of the assembly of Plasmid 1.





This plasmid was constructed to obtain a constant expression of the AlcR protein. As seen in the figure, the constitutive promoter and the alcR were initially present in two different plasmids. They were first digested with the appropriate restriction enzymes, ligated and then transformed into DH5α. The final plasmid construct has a Ampicilin/Carbenicilin resistance. Upon obtaining positive confirmation of the transformants, we proceeded to transform the ligated product into BL21, a protein expression strain.

figure 2
Figure 2: SDS-PAGE gel showing protein expression results. As shown by boxes in the gel, the 27kDa rfp protein can be clearly seen. However, no proteins can be seen at the 98kDa region where the AlcR protein is expected to be present.









After successfully obtaining transformants with the BL21 strain, we proceeded to overexpress the protein and attempted to isolate and purify the 98kDa AlcR protein by SDS-PAGE. Unfortunately, after several attempts, we failed to see any visible protein band on the SDS-PAGE. This could be due to many factors, one of them being that the amount of expressed protein is not enough to be visualised on a SDS-PAGE.

Plasmid 2

figure3
Figure 3: Schematic representation of the assembly of Plasmid 2.





This plasmid is crucial in proving that our mechanism works. It consists of the alcA promoter, which has the AlcR binding site, and chromoproteins to produce a visible colour change. Similar to Plasmid 1, the constitutive promoter and the chromoproteins were initially present in two different plasmids. They were first digested with the appropriate restriction enzymes, ligated and then transformed into DH5α. The final plasmid construct has a Chloramphenicol resistance.


Co-Transformation

In order to prove that our model works, we co-transformed plasmid 1 and plasmid 2 into DH5α and plated them onto a plate containing both Chloramphenicol and Carbenicilin antibiotics. Successful colonies were subjected to quantification using our FLUOstar Image plate reader.


Quantification




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