Part:BBa_K3520021:Design

cmcax Flavobacteriia Transcriptional Unit

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 constitutive 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 thorough 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 in 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 formed 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.