Difference between revisions of "Part:BBa K4317117"
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<partinfo>BBa_K4317117 short</partinfo> | <partinfo>BBa_K4317117 short</partinfo> | ||
| − | Our team used two previously registered parts to facilitate the screening process. One is T2A (BBa_K1993019), a self-cleaving peptide, and the other is sfGFP (BBa_I746916). In order to obtain a transformant that effectively expresses and secretes Kscel7A, known as exo-cellobisidase I, in Pichia pastoris, T2A and sfGFP were fused to the back of the KsCel7A coding sequence with the stop codon removed (Our new composite parts : BBa_K4317116; T2A-sfGFP, BBa_K4317117; KsCel7A-T2A-sfGFP). In the figure 1, the recombinant plasmid DNA was transformed into Pichia pastoris through electroporation. | + | Our team used two previously registered parts to facilitate the screening process. One is T2A (BBa_K1993019), a self-cleaving peptide, and the other is sfGFP (BBa_I746916). In order to obtain a transformant that effectively expresses and secretes Kscel7A, known as exo-cellobisidase I, in Pichia pastoris, T2A and sfGFP were fused to the back of the KsCel7A coding sequence with the stop codon removed (Our new composite parts : BBa_K4317116; T2A-sfGFP, BBa_K4317117; KsCel7A-T2A-sfGFP). In the figure 1, the recombinant plasmid DNA was transformed into Pichia pastoris through electroporation. Colonies selected in the zeocin-added medium were patched on an MM agar plate containing methanol. After incubation at 30°C for 2 days, use a fluorescent device Figure 2) to find transformants expressing sfGFP (Figure 3). Our new composite parts and methods make it easy to find transformants expressing the target protein in pichia. |
| + | https://static.igem.org/mediawiki/parts/thumb/8/8b/PPICZalpha-KsCel7A-T2A-sfGFP.png/577px-PPICZalpha-KsCel7A-T2A-sfGFP.png | ||
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| + | Figure 1. Plasmid map for expression of KsCel7A fused with T2A-sfGFP | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
| + | https://static.igem.org/mediawiki/parts/thumb/4/43/Blueled.jpg/800px-Blueled.jpg | ||
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| + | Figure 2. Blue LED and plastic emission filter for GFP detection | ||
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| + | https://static.igem.org/mediawiki/parts/b/bd/Pichia_t2a.jpg | ||
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| + | Figure 3. Pichia transformants expressing sfGFP. | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K4317117 SequenceAndFeatures</partinfo> | <partinfo>BBa_K4317117 SequenceAndFeatures</partinfo> | ||
Latest revision as of 07:24, 12 October 2022
KsCel7A-T2A-sfGFP
Our team used two previously registered parts to facilitate the screening process. One is T2A (BBa_K1993019), a self-cleaving peptide, and the other is sfGFP (BBa_I746916). In order to obtain a transformant that effectively expresses and secretes Kscel7A, known as exo-cellobisidase I, in Pichia pastoris, T2A and sfGFP were fused to the back of the KsCel7A coding sequence with the stop codon removed (Our new composite parts : BBa_K4317116; T2A-sfGFP, BBa_K4317117; KsCel7A-T2A-sfGFP). In the figure 1, the recombinant plasmid DNA was transformed into Pichia pastoris through electroporation. Colonies selected in the zeocin-added medium were patched on an MM agar plate containing methanol. After incubation at 30°C for 2 days, use a fluorescent device Figure 2) to find transformants expressing sfGFP (Figure 3). Our new composite parts and methods make it easy to find transformants expressing the target protein in pichia.
Figure 1. Plasmid map for expression of KsCel7A fused with T2A-sfGFP
Usage and Biology
Figure 2. Blue LED and plastic emission filter for GFP detection
Figure 3. Pichia transformants expressing sfGFP.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1593
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1665