Difference between revisions of "Team:Ruia-Mumbai/Basic Part"

 
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<h1 style="text-align:center; color:#d80505 ;">BASIC PART</h1><hr>
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Basic Parts:
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<strong>Basic Parts:<a href="http://parts.igem.org/Part:BBa_K2749017">BBa_K2749017</a></strong>
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</div>
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The part codes for the enzyme catechol-2,3-dioxygenase which cleaves the oxidative ring of catechol to form 2-hydroxymuconate semialdehyde. <br>
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It also contains a 6X His-tag which is a useful marker for purification and identification of the protein of interest.
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            &nbsp;
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BBa_K2749017
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xylE
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            &nbsp;
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The part codes for the enzyme catechol-2,3-dioxygenase which cleaves the oxidative ring of catechol to form 2-hydroxymuconate semialdehyde. It also contains a 6X His-tag which is a useful marker for purification and identification of the protein of interest.
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Structure:
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<img src="https://static.igem.org/mediawiki/2018/0/09/T--Ruia-Mumbai--XylE3D.png">
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<img src="https://static.igem.org/mediawiki/2018/6/6c/T--Ruia-Mumbai--composite_part.png" class="image3"><br>
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Fig1: 3D-Structure of enzyme catechol-2,3-dioxygenase
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<img src="https://static.igem.org/mediawiki/2018/6/6c/T--Ruia-Mumbai--composite_part.png"><br>
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Reaction Image taken from paper: https://www.ingentaconnect.com/contentone/scs/chimia<br>
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<a href="https://www.ingentaconnect.com/contentone/scs/chimia/2017/00000071/00000010/art00015?crawler=true&mimetype=application/pdf">Reaction Image Source</a>
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/2017/00000071/00000010/art00015?crawler=true&mimetype=application/pdf
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<img src="https://static.igem.org/mediawiki/2018/3/34/T--Ruia-Mumbai--xylE_character.png"><br>
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Fig2: Construct of the enzyme module
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<strong>3D Structure of enzyme catechol-2,3-dioxygenase</strong>
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Characterisation:
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<img src="https://static.igem.org/mediawiki/2018/0/09/T--Ruia-Mumbai--XylE3D.png" class="image1">
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SDS-PAGE analysis by Coomassie Brilliant Blue staining-
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Overnight culture of the clones were spun down and the pellet was used for analysis of the proteinic enzyme.
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<img src="https://static.igem.org/mediawiki/2018/8/83/T--Ruia-Mumbai--image2-fullgel.png">
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Fig 3: SDS-PAGE analysis by Coomassie Brilliant Blue staining
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The dark protein bands are observed slightly above the 33 kDa ladder which is equal to the expected size of the enzyme catechol-2,3-dioxygenase of 35 kDa. {ref.https://www.ncbi.nlm.nih.gov/pubmed/8713131}
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2-HMS Assay:
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<strong> Construct of the our enzyme module</strong>
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<img src="https://static.igem.org/mediawiki/2018/3/34/T--Ruia-Mumbai--xylE_character.png" class="image2" ><br>
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In this assay, 2-Hydroxymuconate Semialdehyde (2-HMS) is detected, which is the degradation product of catechol, catalysed by enzyme catechol-2,3-dioxygenase (xylE). This product has a characteristic λmax at 380 nm. Here in this assay, we used 415 nm and a range of catechol concentration (0.1 - 0.4 mM) for 2-HMS detection at intervals of 5 mins where total volume of test was 100 uL with the prepared culture suspension (18-24 hrs old) of 0.5 OD.
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pBAD_xylE with Arabinose as substrate (pBAD is a arabinose induced promoter)
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Absorbance readings at 415 nm at different time intervals for range of catechol from 0.1 mM to 0.4 mM
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<img src="img src="https://static.igem.org/mediawiki/2018/5/5e/T--Ruia-Mumbai--table2.png">">
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<img src="https://static.igem.org/mediawiki/2018/7/7e/T--Ruia-Mumbai--graph_2_ara.PNG">
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<p>From the above 3D graphs of Absorbance v/s Time of the reaction system, with respect to increasing concentration of catechol in the range of 0.1 mM to 0.4 mM, it can be interpreted that-
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<ul>
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<li>With advancement in time, the absorbance of the system increases.</li>
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<li>With increasing catechol concentration, the absorbance value increases till 0.35 mM catechol concentration.</li>
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<li>After the concentration of 0.35 mM of catechol, the absorbance readings are found to decrease, which can be suggested from the fact that catechol at high concentrations is toxic for the cell growth.</li>
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{{Ruia-Mumbai/Footer}}
 
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Latest revision as of 17:03, 7 December 2018

 
 


BASIC PART

 
Basic Parts:BBa_K2749017
The part codes for the enzyme catechol-2,3-dioxygenase which cleaves the oxidative ring of catechol to form 2-hydroxymuconate semialdehyde.
It also contains a 6X His-tag which is a useful marker for purification and identification of the protein of interest.
 
 

Reaction Image Source
 
3D Structure of enzyme catechol-2,3-dioxygenase
 
Construct of the our enzyme module

 

Our co-ordinates:

L .Nappo Road, Matunga
Dadar East,Mumbai,
Maharashtra 400019,
India

catechewingcoli@gmail.com

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