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

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Basic Parts:
 
Basic Parts:
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BBa_K2749017
 
BBa_K2749017
 
xylE
 
xylE
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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.
 
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:
 
Structure:
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<img src="https://static.igem.org/mediawiki/2018/0/09/T--Ruia-Mumbai--XylE3D.png">
 
<img src="https://static.igem.org/mediawiki/2018/0/09/T--Ruia-Mumbai--XylE3D.png">
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Fig1: 3D-Structure of enzyme catechol-2,3-dioxygenase  
 
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>
 
<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>
 
Reaction Image taken from paper: https://www.ingentaconnect.com/contentone/scs/chimia<br>
 
/2017/00000071/00000010/art00015?crawler=true&mimetype=application/pdf
 
/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>
 
<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
 
Fig2: Construct of the enzyme module
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Characterisation:
 
Characterisation:
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SDS-PAGE analysis by Coomassie Brilliant Blue staining-
 
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.
 
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">
 
<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
 
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}
 
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:
 
2-HMS Assay:
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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.
 
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)
 
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
 
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="https://static.igem.org/mediawiki/2018/7/7e/T--Ruia-Mumbai--graph_2_ara.PNG">
 
<img src="https://static.igem.org/mediawiki/2018/7/7e/T--Ruia-Mumbai--graph_2_ara.PNG">
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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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<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-
 
 
<ul>
 
<ul>
 
<li>With advancement in time, the absorbance of the system increases.</li>
 
<li>With advancement in time, the absorbance of the system increases.</li>
 
<li>With increasing catechol concentration, the absorbance value increases till 0.35 mM catechol concentration.</li>
 
<li>With increasing catechol concentration, the absorbance value increases till 0.35 mM catechol concentration.</li>
 
<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>
 
<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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Revision as of 13:48, 16 October 2018

 
Basic Parts:
 
BBa_K2749017 xylE
 
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.
 
Structure:
 
 
Fig1: 3D-Structure of enzyme catechol-2,3-dioxygenase
 

 
Reaction Image taken from paper: https://www.ingentaconnect.com/contentone/scs/chimia
/2017/00000071/00000010/art00015?crawler=true&mimetype=application/pdf
 

 
Fig2: Construct of the enzyme module
 
Characterisation:
 
SDS-PAGE analysis by Coomassie Brilliant Blue staining-
 
Overnight culture of the clones were spun down and the pellet was used for analysis of the proteinic enzyme.
 
 
Fig 3: SDS-PAGE analysis by Coomassie Brilliant Blue staining
 
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}
 
2-HMS Assay:
 
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.
 
pBAD_xylE with Arabinose as substrate (pBAD is a arabinose induced promoter)
 
Absorbance readings at 415 nm at different time intervals for range of catechol from 0.1 mM to 0.4 mM
 
 
 
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-
 
  • With advancement in time, the absorbance of the system increases.
  • With increasing catechol concentration, the absorbance value increases till 0.35 mM catechol concentration.
  • 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.
 

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