Difference between revisions of "Part:BBa K3520013:Design"
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==Promoter Coice== | ==Promoter Coice== | ||
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The ompA promoter is a constituent and strong Flavobacterial promoter. Our project's goal is the large production of structurally coloured cellulose, so a strong promoter is deemed necessary. | The ompA promoter is a constituent and strong Flavobacterial promoter. Our project's goal is the large production of structurally coloured cellulose, so a strong promoter is deemed necessary. | ||
<br> | <br> | ||
==RBS Choice== | ==RBS Choice== | ||
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This RBS is strong, and Flavobacteria specific. | This RBS is strong, and Flavobacteria specific. | ||
<br> | <br> | ||
==Terminator Choice== | ==Terminator Choice== | ||
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This strong terminator sequence is chosen based on its universality and its efficient characterisation. | This strong terminator sequence is chosen based on its universality and its efficient characterisation. | ||
<br><br> | <br><br> | ||
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=Assembly= | =Assembly= | ||
The assembly method used to create this TU is TypeIIS. | The assembly method used to create this TU is TypeIIS. | ||
| + | <br> | ||
[[File:T--Athens--Level_0_design.png|800px|thumb|center|Figure 1: Type IIS design with RFC10 primers for TU parts.]] | [[File:T--Athens--Level_0_design.png|800px|thumb|center|Figure 1: Type IIS design with RFC10 primers for TU parts.]] | ||
| + | <br> | ||
==Why== | ==Why== | ||
| − | |||
We opted for this method of assembly based on many factors. For one, the scarless ligations lowered the level of uncertainty and noise on our construct. Also, should we be forced to order the CDS in several parts, due to its big size, we could easily ligate the parts, with no indels or stop codon insertions. | We opted for this method of assembly based on many factors. For one, the scarless ligations lowered the level of uncertainty and noise on our construct. Also, should we be forced to order the CDS in several parts, due to its big size, we could easily ligate the parts, with no indels or stop codon insertions. | ||
<br> | <br> | ||
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<br> | <br> | ||
==How== | ==How== | ||
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Our ordered DNA (level 0) was flanked both on the 5' and the 3' end with specific sequences. These, among other things, contained BsaI sites which, upon restriction digestion would leave specific overhangs that would ensure ligation in the right order. A PCR reaction on this newly fromed level 1 Transcriptional Unit would introduce the specific SapI level 1 sites, in order for the second step of ligation to occur, giving rise to the level 2 Multi Transcriptional Unit. | Our ordered DNA (level 0) was flanked both on the 5' and the 3' end with specific sequences. These, among other things, contained BsaI sites which, upon restriction digestion would leave specific overhangs that would ensure ligation in the right order. A PCR reaction on this newly fromed level 1 Transcriptional Unit would introduce the specific SapI level 1 sites, in order for the second step of ligation to occur, giving rise to the level 2 Multi Transcriptional Unit. | ||
<br> | <br> | ||
Revision as of 02:00, 28 October 2020
bcsA-Bacterial Cellulose Synthase A TU
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
DESIGN CONSIDERATIONS
Promoter Coice
The ompA promoter is a constituent and strong Flavobacterial promoter. Our project's goal is the large production of structurally coloured cellulose, so a strong promoter is deemed necessary.
RBS Choice
This RBS is strong, and Flavobacteria specific.
Terminator Choice
This strong terminator sequence is chosen based on its universality and its efficient characterisation.
Assembly
The assembly method used to create this TU is TypeIIS.
Why
We opted for this method of assembly based on many factors. For one, the scarless ligations lowered the level of uncertainty and noise on our construct. Also, should we be forced to order the CDS in several parts, due to its big size, we could easily ligate the parts, with no indels or stop codon insertions.
Second, a factor that contributed largely to our choice was the single-pot, single-reaction nature of the reaction. Our project, as many iGEM projects, contained a large number of assemblies, and thus such a reaction could save time, recourses, and failed experiments.
How
Our ordered DNA (level 0) was flanked both on the 5' and the 3' end with specific sequences. These, among other things, contained BsaI sites which, upon restriction digestion would leave specific overhangs that would ensure ligation in the right order. A PCR reaction on this newly fromed level 1 Transcriptional Unit would introduce the specific SapI level 1 sites, in order for the second step of ligation to occur, giving rise to the level 2 Multi Transcriptional Unit.