Difference between revisions of "Part:BBa K3629012"
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===Usage and Biology=== | ===Usage and Biology=== | ||
+ | |||
+ | Fully functional cellulase is composed of: | ||
+ | |||
+ | <ol> | ||
+ | <li>Endoglucanases (EG) which randomly cleave internal beta-bonds of cellulose polymers to make them shorter </li> | ||
+ | <li>Cellobiohydrolases (CBH or exoglucanases) which cleave the shorter polymers to make cellobiose </li> | ||
+ | <ul> | ||
+ | <li>CBHI= Acts on reducing end of sugar molecule </li> | ||
+ | <li>CBHII= Acts on non-reducing end of sugar molecule </li> | ||
+ | </ul> | ||
+ | <li>Beta-glucosidases (BGS) which cleave the cellobiose disaccharide to free glucose units </li> | ||
+ | </ol> | ||
+ | |||
+ | 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 [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> 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=== | ===Design=== |
Revision as of 01:19, 27 October 2020
T. reesei CBHII expression construct with gibson homology sequences and FLAG tag
Usage and Biology
Fully functional cellulase is composed of:
- Endoglucanases (EG) which randomly cleave internal beta-bonds of cellulose polymers to make them shorter
- 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
- 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
The native signal peptide from T. reesei was removed so it would not interfere with the fused Lip2 secretion tag native to Y. lipolytica.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 2737
Illegal EcoRI site found at 2561 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE WITH RFC[23]Unknown
- 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]