Difference between revisions of "Part:BBa K3520013:Design"
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=Assembly=
 
=Assembly=
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The assembly method that used to create this TU is TypeIIS.
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The assembly method used to create this TU is TypeIIS.
  
 
[[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.]]
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==Why==
 
==Why==
 
<br>
 
<br>
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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. Aslo, 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>
−
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 failex experiments.
+
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.
 
<br>
 
<br>
 
==How==
 
==How==
 
<br>
 
<br>
−
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 whcih, upon restriction digestion would leave specific overhangs that would ensure ligation in the right order. This level 1 Transcriptional Unit would then be
+
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 01:59, 28 October 2020


bcsA-Bacterial Cellulose Synthase A TU


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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.

Figure 1: Type IIS design with RFC10 primers for TU parts.

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.