Difference between revisions of "Part:BBa K3895016"
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<partinfo>BBa_K3895016 short</partinfo> | <partinfo>BBa_K3895016 short</partinfo> | ||
==Introduction== | ==Introduction== | ||
| − | This composite part is an optimized version of KERA that can be constructed into pSB1C3 plasmid with the highest protein expression in E.coli BL21(DE3). The KERA (<partinfo>K1717171</partinfo>) is a basic part comprised from a synthesized KERA protein coding sequence. The gene can be expressed in cells by looking for clear areas of cell growth on AGAR plates of skimmed milk, and by observing keratin-containing substrates (feathers, hair, chemosynthetic keratin, etc.) growing with cells. | + | This composite part is an optimized version of KERA that can be constructed into pSB1C3 plasmid with the highest protein expression in E.coli BL21(DE3). The KERA (<partinfo>K1717171</partinfo>) is a basic part comprised from a synthesized KERA protein coding sequence. The gene can be expressed in cells by looking for clear areas of cell growth on AGAR plates of skimmed milk, and by observing keratin-containing substrates (feathers, hair, chemosynthetic keratin, etc.) growing with cells. Detailed optimization data can be checked here: https://2021.igem.org/wiki/images/0/04/T--SZ_SHD--dataset.csv |
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==Plasmid Construction== | ==Plasmid Construction== | ||
| − | [[File:T--SZ SHD--kera.png|400px|center]] | + | [[File:T--SZ SHD--kera.png|400px|center]]<br> |
| + | '''Figure 1.''' The construction of optimized KERA on pSB1C3 backbone, with 6xHis-tag for purification. The combination of promoter, RBS, and terminator is with the best performance in expression. | ||
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==Experiment details== | ==Experiment details== | ||
• Throughput: 2500<br> | • Throughput: 2500<br> | ||
Latest revision as of 15:44, 21 October 2021
Optimized keratinase KerA
Introduction
This composite part is an optimized version of KERA that can be constructed into pSB1C3 plasmid with the highest protein expression in E.coli BL21(DE3). The KERA (BBa_K1717171) is a basic part comprised from a synthesized KERA protein coding sequence. The gene can be expressed in cells by looking for clear areas of cell growth on AGAR plates of skimmed milk, and by observing keratin-containing substrates (feathers, hair, chemosynthetic keratin, etc.) growing with cells. Detailed optimization data can be checked here: https://2021.igem.org/wiki/images/0/04/T--SZ_SHD--dataset.csv
Plasmid Construction
Figure 1. The construction of optimized KERA on pSB1C3 backbone, with 6xHis-tag for purification. The combination of promoter, RBS, and terminator is with the best performance in expression.
Experiment details
• Throughput: 2500
• Organism: E. coli
• Strain type: BL21(DE3)
• Temperature: 37°C
• Media: 250 ml LB
• Period of time: 16 hours
• Plasmid backbone: pSB1C3
A. Vec platform was used to test the expression of KERA in more than 2000 random expression vectors. Different regulatory elements used in the vector can result in nearly 10 orders of magnitude difference in KERA expression.
Figure 2. The data plot of KERA optimization. Within more than 2000 protein yields, the final relative unit of the expression vector is about 10 orders of magnitude higher than before.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 326
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 587
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1369
Illegal SapI site found at 957
Illegal SapI site found at 1260