Device

Part:BBa_K3629012

Designed by: Sravya Kakumanu   Group: iGEM20_Calgary   (2020-10-18)
Revision as of 05:18, 27 October 2020 by Sravyakakumanu (Talk | contribs) (→‎Design)


T. reesei CBHII expression construct with gibson homology sequences and FLAG 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 creating 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

Table 1. Creation of three Yarrowia lipolytica strains each secreting one class of cellulase enzymes. The parts in the first column should be individually digested by the corresponding enzymes in the second column. All of the digested products should then be put in the same tube with the Gibson reagents to create the resulting plasmid/gene cassette in column three.

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

This expression construct was designed to be assembled with other expression constructs from our collection via Gibson Assembly:



These expression constructs can be assembled together in different combinations creating larger gene cassettes (figure 1). Over 20 different gene cassettes can be formed using these parts. The full tables outlining the assembly of these different cassettes can be found here. However, the most essential part is the Nourseothricin resistance expression construct [1], which functions as a destination vector when cloned into a plasmid (Figure 1). Click here to watch a short animation on how this works.

Figure 1.


To create the three strains of Y. lipolytica that could be co-cultured for full cellulase activity, the individual expression constructs must be digested as per table 1. The digestion products can then be put together and mixed with the Gibson reagents for the final gene cassettes to form (table 1). Once the gene cassettes are assembled they can be linearized with NotI and transformed into 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 2737
    Illegal EcoRI site found at 2561
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
    Illegal EcoRI site found at 2561
    Illegal NheI site found at 75
    Illegal SpeI site found at 2738
    Illegal PstI site found at 2752
    Illegal NotI site found at 7
    Illegal NotI site found at 2745
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1
    Illegal EcoRI site found at 2561
    Illegal BamHI site found at 2686
    Illegal XhoI site found at 124
    Illegal XhoI site found at 1198
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 2738
    Illegal EcoRI site found at 2561
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1
    Illegal EcoRI site found at 2561
    Illegal XbaI site found at 16
    Illegal SpeI site found at 2738
    Illegal PstI site found at 2752
    Illegal NgoMIV site found at 1458
  • 1000
    COMPATIBLE WITH RFC[1000]


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