Difference between revisions of "Part:BBa K4205111:Experience"
 
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<!-- DON'T DELETE --><partinfo>BBa_K4205111 EndReviews</partinfo>
 
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<p>Since the 3D structure of LldR in our chassis, Escherichia coli Nissle 1917, was not found in PDB, we used SWISS-MODEL for homology modeling to predict the 3D structure of LldR.</p>
 
<p>Since the 3D structure of LldR in our chassis, Escherichia coli Nissle 1917, was not found in PDB, we used SWISS-MODEL for homology modeling to predict the 3D structure of LldR.</p>
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[[File:lldR SWISS.png|300px|center|thumb|left|Predicted 3D structure of LldR]]<br>
 
<p>With the help of AutoDock, we simulated the docking of LldR and L-lactate. The resulting figure is presented below. The docking sites are colored yellow.</p>
 
<p>With the help of AutoDock, we simulated the docking of LldR and L-lactate. The resulting figure is presented below. The docking sites are colored yellow.</p>
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[[File:lldr and lactate.png|300px|center|thumb|left|Docking of LldR and L-lactate]]<br>
 
<p>We constructed the ALPaGA-lacZ plasmid and transformed it into ''Escherichia coli'' DH5α. To prove the success of our transformation, we designed primers, performed plasmid PCR and colony PCR to amplify the LldR gene, and ran the agarose gal.</p>
 
<p>We constructed the ALPaGA-lacZ plasmid and transformed it into ''Escherichia coli'' DH5α. To prove the success of our transformation, we designed primers, performed plasmid PCR and colony PCR to amplify the LldR gene, and ran the agarose gal.</p>
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<p>As shown in the figure below, after plasmid PCR and colony PCR, the sample from the transformed DH5α both ran out bands near 750 bp, which was consistent with the target band (777 bp). This further indicated that our plasmid transformation was successful.</p>
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[[File:4-LldRPCR.png|700px|center|thumb|left|Proof of the successful transformation.(A) plasmid PCR. Lanes 1-2: PCR sample form transformed DH5α. Lanes 3-4: PCR sample form untransformed DH5α.  
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[[File:lldR SWISS.png|300px|thumb|left|Predicted 3D structure of LldR]]
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[[File:lldr and lactate.png|300px|thumb|left|Docking of LldR and L-lactate]]
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[[File:4-LldRPCR.png|700px|thumb|left|Proof of the successful transformation.(A) plasmid PCR. Lanes 1-2: PCR sample form transformed DH5α. Lanes 3-4: PCR sample form untransformed DH5α.  
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(B) colony PCR. Lanes 1-4: PCR sample form transformed DH5α. Lanes 5-8: PCR sample form untransformed DH5α.
 
(B) colony PCR. Lanes 1-4: PCR sample form transformed DH5α. Lanes 5-8: PCR sample form untransformed DH5α.
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<p>As shown in the figure above, after plasmid PCR and colony PCR, the sample from the transformed DH5α both ran out bands near 750 bp, which was consistent with the target band (777 bp). This further indicated that our plasmid transformation was successful.</p>

Latest revision as of 11:11, 13 October 2022


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UNIQeb7c6a0f099f2089-partinfo-00000000-QINU UNIQeb7c6a0f099f2089-partinfo-00000001-QINU

Since the 3D structure of LldR in our chassis, Escherichia coli Nissle 1917, was not found in PDB, we used SWISS-MODEL for homology modeling to predict the 3D structure of LldR.

Predicted 3D structure of LldR

With the help of AutoDock, we simulated the docking of LldR and L-lactate. The resulting figure is presented below. The docking sites are colored yellow.

Docking of LldR and L-lactate

We constructed the ALPaGA-lacZ plasmid and transformed it into Escherichia coli DH5α. To prove the success of our transformation, we designed primers, performed plasmid PCR and colony PCR to amplify the LldR gene, and ran the agarose gal.

Proof of the successful transformation.(A) plasmid PCR. Lanes 1-2: PCR sample form transformed DH5α. Lanes 3-4: PCR sample form untransformed DH5α. (B) colony PCR. Lanes 1-4: PCR sample form transformed DH5α. Lanes 5-8: PCR sample form untransformed DH5α.

As shown in the figure above, after plasmid PCR and colony PCR, the sample from the transformed DH5α both ran out bands near 750 bp, which was consistent with the target band (777 bp). This further indicated that our plasmid transformation was successful.