Difference between revisions of "Part:BBa K3629012"
(→Design) |
(→Design) |
||
Line 39: | Line 39: | ||
</ul> | </ul> | ||
− | |||
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 [https://2020.igem.org/Team:Calgary/Appendix#part-design here.] However, the most essential part is the Nourseothricin resistance expression construct [https://parts.igem.org/Part:BBa_K3629015 BBa_K3629015](BBa_K3629015)], which functions as a destination vector when cloned into a plasmid (Figure 1). Click [https://2020.igem.org/wiki/images/e/e4/T--Calgary--partcollectionvideo1.mp4 here] to watch a short animation on how this works. | 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 [https://2020.igem.org/Team:Calgary/Appendix#part-design here.] However, the most essential part is the Nourseothricin resistance expression construct [https://parts.igem.org/Part:BBa_K3629015 BBa_K3629015](BBa_K3629015)], which functions as a destination vector when cloned into a plasmid (Figure 1). Click [https://2020.igem.org/wiki/images/e/e4/T--Calgary--partcollectionvideo1.mp4 here] to watch a short animation on how this works. | ||
+ | |||
+ | [[Image:T--Calgary--Gibsonstuff.png|400px|center|Figure 1.]] | ||
Revision as of 05:15, 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.
This expression construct was designed to be assembled with other expression constructs from our collection via Gibson Assembly:
- BBa_K3629015= Nourseothricin resistance expression construct
- BBa_K3629013= Modified P. funiculosum CBHI expression construct
- BBa_K3629014= N. crassa CBHI expression construct
- BBa_K3629012= TrCBHII expression construct
- BBa_K3629016= Modified T. reesei EGI expression construct
- BBa_K3629017= T. reesei EGII expression construct
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 BBa_K3629015(BBa_K3629015)], which functions as a destination vector when cloned into a plasmid (Figure 1). Click here to watch a short animation on how this works.
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
- 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]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 2738
Illegal EcoRI site found at 2561 - 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]