Composite

Part:BBa_K4989011:Design

Designed by: Athanasia Arampatzi   Group: iGEM23_Thrace   (2023-10-12)
Revision as of 13:52, 12 October 2023 by Atharab (Talk | contribs) (→‎Design Notes)


Complete butyrate producing gene cluster


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1143
    Illegal BglII site found at 6852
    Illegal BamHI site found at 1008
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 291
    Illegal NgoMIV site found at 2703
    Illegal NgoMIV site found at 6067
    Illegal AgeI site found at 1032
    Illegal AgeI site found at 1066
    Illegal AgeI site found at 5423
    Illegal AgeI site found at 7026
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

In this section, we will shortly analyze the re-design process of our part. The extensions of this process and the more detailed handling are analyzed in our team's New Improved Part Wiki section (see at the end of the page). The re-design project was performed on 3 separate levels:

A. On a sequence level B. On a regulatory level C. On a structural level

On a sequence level, we needed to change the whole DNA makeup of the genes and codon-optimize them to be properly expressed in the corresponding hosts. Also, we performed an in-silico optimization of the sequence to not include any restriction sites of the endonucleases that we intended to use.

On a regulatory level, we added some very crucial regulatory elements to ensure the expression of the enzymes. We included the P32 promoter with its RBS for the expression to be compatible with our target host Lactobacillus rhamnosus GG. Also, we added at the of the part a terminator sequence specifically designed for Lactobacillus spp.In this way, we ensured our transcriptional termination. Lastly, we included in-between the coding sequences Ribosome Binding Sites, for one transcriptional mRNA to be produced by all the target enzymes of the pathway.

On a structural level, we maintained the order of the genes and we added, upon prompt from our dry lab bioinformatic analysis, the butyryl-CoA:acetyl-CoA transferase that catalyzes the last step of the butyrate production.

Our new improved part has the characteristic of a whole constructed transcriptional cassette. However, we have marginalized all the basic parts (components) for the P32 promoter and the terminator to be removed, always based on the regulatory feature that the cloning vector has.

Source

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References