Difference between revisions of "Part:BBa K4368000"
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<partinfo>BBa_K4368000 short</partinfo>
 
<partinfo>BBa_K4368000 short</partinfo>
  
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==Description==
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''CenA'' encodes for the endoglucanase gene of ''Cellulomonas fimi'' (<partinfo>BBa_K118023</partinfo>). This enzyme is responsible of the degradation of cellulose working coordinated with the genes ''cex'' 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:cen.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|]]
  
 
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<span class='h3bb'>Sequence and Features</span>
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==<span class='h3bb'>Sequence and Features</span>==
 
<partinfo>BBa_K4368000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4368000 SequenceAndFeatures</partinfo>
  
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==Contribution ==
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*'''Group:''' [https://2022.igem.wiki/uma-malaga/index.html UMA_MALAGA]
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*'''Author:''' Molina Calvo, Alonso
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
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Latest revision as of 08:35, 11 October 2022

pcstA + rbs + yebF + cenA + terminator

Description

CenA encodes for the endoglucanase gene of Cellulomonas fimi (BBa_K118023). This enzyme is responsible of the degradation of cellulose working coordinated with the genes cex 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

Cen.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


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 554
    Illegal NotI site found at 1698
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 268
    Illegal BglII site found at 1611
    Illegal BamHI site found at 747
    Illegal XhoI site found at 1109
    Illegal XhoI site found at 1358
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 889
    Illegal NgoMIV site found at 1814
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 793

Contribution