Difference between revisions of "Part:BBa K3629014"
(→‎Usage and Biology)
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__TOC__
 
__TOC__
 
===Usage and Biology===
 
===Usage and Biology===
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Fully functional cellulase is composed of:
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 +
<ol>
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<li>Endoglucanases (EG) which randomly cleave internal beta-bonds of cellulose polymers to make them shorter </li>
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<li>Cellobiohydrolases (CBH or exoglucanases) which cleave the shorter polymers to make cellobiose </li>
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<ul>
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<li>CBHI= Acts on reducing end of sugar molecule </li>
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<li>CBHII= Acts on non-reducing end of sugar molecule </li>
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</ul>
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<li>Beta-glucosidases (BGS) which cleave the cellobiose disaccharide to free glucose units </li>
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</ol>
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These proteins must  be in the correct proportions to each other to efficiently degrade cellulose.
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This expression construct can be used in the assembly of a CBH gene cassette containing Modified PfCBHI [https://parts.igem.org/Part:BBa_K3629013 (BBa_K3629013)], NcCBHI [https://parts.igem.org/Part:BBa_K3629014 (BBa_K3629014)], and TrCBHII [https://parts.igem.org/Part:BBa_K3629012 (BBa_K3629012)]. This gene cassette is then intended to be transformed into <i>Y. lipolytica</i> to create a CBH- producing strain. This strain should then be co-cultured with two other strains with a EG or BGS gene cassette. The three strains together will be able to survive on cellulose media.
  
 
===Design===
 
===Design===

Revision as of 06:07, 27 October 2020


N. crassa CBHI expression construct with gibson homology sequences and Myc tag

Usage and Biology

Fully functional cellulase is composed of:

  1. Endoglucanases (EG) which randomly cleave internal beta-bonds of cellulose polymers to make them shorter
  2. Cellobiohydrolases (CBH or exoglucanases) which cleave the shorter polymers to make cellobiose
    • CBHI= Acts on reducing end of sugar molecule
    • CBHII= Acts on non-reducing end of sugar molecule
  3. Beta-glucosidases (BGS) which cleave the cellobiose disaccharide to free glucose units

These proteins must be in the correct proportions to each other to efficiently degrade cellulose.

This expression construct can be used in the assembly of a CBH gene cassette containing Modified PfCBHI (BBa_K3629013), NcCBHI (BBa_K3629014), and TrCBHII (BBa_K3629012). This gene cassette is then intended to be transformed into Y. lipolytica to create a CBH- producing strain. This strain should then be co-cultured with two other strains with a EG or BGS gene cassette. The three strains together will be able to survive on cellulose media.

Design

The native signal peptide from N. crassa was removed so it would not interfere with the fused Lip2 secretion tag native to Y. lipolytica.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 2751
    Illegal EcoRI site found at 1054
    Illegal EcoRI site found at 2570
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
    Illegal EcoRI site found at 1054
    Illegal EcoRI site found at 2570
    Illegal NheI site found at 75
    Illegal SpeI site found at 2752
    Illegal PstI site found at 2766
    Illegal NotI site found at 7
    Illegal NotI site found at 2759
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1
    Illegal EcoRI site found at 1054
    Illegal EcoRI site found at 2570
    Illegal BglII site found at 1343
    Illegal BglII site found at 1703
    Illegal BamHI site found at 2700
    Illegal XhoI site found at 2745
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 2752
    Illegal EcoRI site found at 1054
    Illegal EcoRI site found at 2570
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1
    Illegal EcoRI site found at 1054
    Illegal EcoRI site found at 2570
    Illegal XbaI site found at 16
    Illegal SpeI site found at 2752
    Illegal PstI site found at 2766
    Illegal AgeI site found at 2211
  • 1000
    COMPATIBLE WITH RFC[1000]

There is an EcoRI site within the XRP2 terminator making this part RFC10 incompatible. However, we added the BioBrick prefix and suffix so that the other enzymes (NotI, XbaI, SpeI, and PstI) could be used to clone this part into an iGEM plasmid or another plasmid. This part can also be cloned through RFC1000 assembly.

The promoter (BBa_K3629001) is an intronic promoter, however the wild-type sequence contains BsaI, PstI, and SpeI restriction sites in the promoter section before the intron sequence. Therefore, single-nucleotides changes that match the ones in the functional BBa_K2983050 promoter were made to remove theses sites.