Difference between revisions of "Part:BBa K118022:Experience"
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''Contributed by British_Columbia 2016 iGEM team.''<br><br>
 
''Contributed by British_Columbia 2016 iGEM team.''<br><br>
  
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We have selected CEX protein to be displayed on the surface of <i>Caulobacter crescentus</i> for transformation of lignocellulose into fermentable sugars. The CEX construct from 43 to 443 amino acids position was cloned into rsaA protein in p4A723 plasmid and transformed into <i>Caulobacter crescentus</i>. The cellulase activity was tested in a test with 2,4-dinitrophenylcellobiose(DNPC) substrate, which consists of cellubiose bound to a chromophore. If cellulase activity is present, the release of chromophore can be observed and measured at OD400. CEX displayed on the <i>C.crescentus</i> surface showed significantly higher activity than wild type rsaA. The results of the assay after 12 hours are shown on the Picture.<br>
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We have selected CEX protein to be displayed on the surface of <i>Caulobacter crescentus</i> for transformation of lignocellulose into fermentable sugars. The CEX construct from 43 to 443 amino acids position was cloned into rsaA protein in p4A723 plasmid and transformed into <i>Caulobacter crescentus</i>. The cellulase activity was tested in a test with 2,4-dinitrophenylcellobiose(DNPC) substrate, which consists of cellubiose bound to a chromophore. If cellulase activity is present, the release of chromophore can be observed and measured at OD400. CEX displayed on the <i>C.crescentus</i> surface showed significantly higher activity than wild type rsaA. The results of the assay after 12 hours are shown on the Picture.
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<center>
 
 
[[File:Cex_dnpc.png|200px]]
 
[[File:Cex_dnpc.png|200px]]
  
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===Characterization from iGEM19_CAU_China===
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We linke the cex gene BBa_K118022 into a pET30a(+) backbone, then transforme this plasmid into BL21. After the overnight (about 15h) induction under the condition of 16℃ 0.08 mM, we smash the recombinant E.coli strain with the ultrasonication to get crude enzyme solution, then we measured the enzyme activity by the method of CMC-Na(sodium carboxymethyl cellulose) assey. The result is shown on Fig.3[1].
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==Improvement UMA_MALAGA 2022==
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*'''Group:''' [https://2022.igem.wiki/uma-malaga/index.html UMA_MALAGA]
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*'''Author:''' Molina Calvo, Alonso
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==Description==
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''Cex'' encodes for the exoglucanase gene of ''Cellulomonas fimi'' (<partinfo>BBa_K118022</partinfo>). This enzyme is responsible of the degradation of cellulose working coordinated with the genes ''cenA'' and ''bglX''. In addition, this part includes the composition used by the team, which includes a strong rbs (<partinfo>BBa_B0030</partinfo>), a double terminator (<partinfo>BBa_B0015</partinfo>) as well as a promoter inducible by glucose concentration (<partinfo>BBa_K118011</partinfo>). '''Furthermore, we improve this composite adding a gen encoding a motor protein named ''YebF''''' (<partinfo>BBa_K1610300</partinfo>) that secrete the enzyme out of the ''E. coli'' membrane.
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The gene has been placed under the control of this promoter to build the glucose concentration-based gene regulatory circuit that integrates all our parts.
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==Characterization==
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The expression cassette sequence was digested with EcoRI and PstI enzymes and subsequently ligated with a chloramphenicol resistant plasmid backbone (Cm).
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Transforming bacteria were created with this plasmid and seeded on LB-Agar+Cm plates. After growth, colonies were selected based on their color (white) and DNA extraction was performed using the Promega PureYield Plasmid Miniprep System kit.
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The resulting DNA is used for further digestion with EcoRI and PstI. The digests are then run on a 0.75% agarose gel at 90 mV voltage and constant amperage. BioRad brand RedSafe is used as an intercalating developing agent.
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===Enzyme digestion===
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[[File:cex.png|350px|center|]]
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===Congo red assay===
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A colorimetric assay for the detection of cellulase activity by bacteria transformed by this plasmid was performed. For this purpose, plates with cellulose-rich medium were created and 50 μL of the transformed bacterial suspension were seeded in the center. After 48 hours, it is developed using a 0.1% solution of Congo red. It is left to incubate in agitation at 400 rpm for 15 minutes. After this incubation time, the plates are washed with 1M NaCl solution and the plates are observed. If cellulose degradation has occurred, an orange halo is created around the bacterial zone, while the rest of the plate will appear reddish. This is because congo red differentially stains cellulose and does not stain cellobiose.
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[[File:congo_red.png|350px|center|]]
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 10:50, 13 October 2022

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K118022

Characterization

Edinburgh 2011 conducted two assays, comparing the activity of this part (but under the control of the lac promoter, giving BBa_K523016) to a β-glucosidase (E. coli bglX, also under the control of the lac promoter) on two different substrates:

  • 4-methylumbelliferyl β- D- glucuronide (MUG, left photo). This substrate is a cellobiose analog.
  • 4-methylumbelliferyl β- D- cellobioside (MUC, right photo). This substrate is larger and is more like a cellulose analog.

Both substrates produce a fluorescent product when cleaved. Our plates below show the results of placing cell lysate and cell debris on an MUG plate and an MUC plate. Present on both plates are:

  • Left side of plate: lysate/debris from JM109 expressing bglX, BBa_K523014
  • Right side of plate: lysate/debris from JM109 expressing cex, BBa_K523016
  • Bottom of plate: lysate/debris from JM109 cells
BglX-MuG.jpg     Cex-MuC.jpg
MUG assay. bglX on left, cex on right.     MUC assay. bglX on left, cex on right.

As can be seen, cex is much better at degrading MUC.


Further Characterization of BBa_K118022 on S-layer of Caulobacter crescentus

Contributed by British_Columbia 2016 iGEM team.

We have selected CEX protein to be displayed on the surface of Caulobacter crescentus for transformation of lignocellulose into fermentable sugars. The CEX construct from 43 to 443 amino acids position was cloned into rsaA protein in p4A723 plasmid and transformed into Caulobacter crescentus. The cellulase activity was tested in a test with 2,4-dinitrophenylcellobiose(DNPC) substrate, which consists of cellubiose bound to a chromophore. If cellulase activity is present, the release of chromophore can be observed and measured at OD400. CEX displayed on the C.crescentus surface showed significantly higher activity than wild type rsaA. The results of the assay after 12 hours are shown on the Picture.

Cex dnpc.png


Improvement UMA_MALAGA 2022

Description

Cex encodes for the exoglucanase gene of Cellulomonas fimi (BBa_K118022). This enzyme is responsible of the degradation of cellulose working coordinated with the genes cenA and bglX. In addition, this part includes the composition used by the team, which includes a strong rbs (BBa_B0030), a double terminator (BBa_B0015) as well as a promoter inducible by glucose concentration (BBa_K118011). Furthermore, we improve this composite adding a gen encoding a motor protein named YebF (BBa_K1610300) that secrete the enzyme out of the E. coli membrane. The gene has been placed under the control of this promoter to build the glucose concentration-based gene regulatory circuit that integrates all our parts.

Characterization

The expression cassette sequence was digested with EcoRI and PstI enzymes and subsequently ligated with a chloramphenicol resistant plasmid backbone (Cm). Transforming bacteria were created with this plasmid and seeded on LB-Agar+Cm plates. After growth, colonies were selected based on their color (white) and DNA extraction was performed using the Promega PureYield Plasmid Miniprep System kit. The resulting DNA is used for further digestion with EcoRI and PstI. The digests are then run on a 0.75% agarose gel at 90 mV voltage and constant amperage. BioRad brand RedSafe is used as an intercalating developing agent.

Enzyme digestion

Cex.png

Congo red assay

A colorimetric assay for the detection of cellulase activity by bacteria transformed by this plasmid was performed. For this purpose, plates with cellulose-rich medium were created and 50 μL of the transformed bacterial suspension were seeded in the center. After 48 hours, it is developed using a 0.1% solution of Congo red. It is left to incubate in agitation at 400 rpm for 15 minutes. After this incubation time, the plates are washed with 1M NaCl solution and the plates are observed. If cellulose degradation has occurred, an orange halo is created around the bacterial zone, while the rest of the plate will appear reddish. This is because congo red differentially stains cellulose and does not stain cellobiose.

Congo red.png

User Reviews

UNIQ47daf68f85552307-partinfo-00000008-QINU UNIQ47daf68f85552307-partinfo-00000009-QINU