Protein_Domain

Part:BBa_K3629003

Designed by: Sravya Kakumanu   Group: iGEM20_Calgary   (2020-10-10)
Revision as of 00:54, 27 October 2020 by Sravyakakumanu (Talk | contribs) (→‎Design)


Yarrowia lipolytica TEF1 promoter with first half of Lip2 signal peptide

TEF1 promoter from Yarrowia lipolytica and the first 42nts (14 codons) of the Lip2 signal peptide (BBa_K1592000)from Y. lipolytica.


Usage and Biology

The part contains wild-type TEF1 promoter (nucleotides 1227374 to 1226969) from Yarrowia lipolytica W29 chromosome C (GenBank Accession #CP028450.1) matching BBa_K2117000, and the first 42nts (14 codons) of the Lip2 signal peptide from Y. lipolytica (BBa_K1592000)

Design

This promoter is composed of the wild-type TEF1 promoter (BBa_K2117000) and the first part of the Lip2 signal peptide (BBa_K1592000). It was designed to be exchanged with the TEFin promoter (BBa_K3629001) placed in our other expression constructs with a Lip2 signal peptide in case the TEFin promoter was not working properly.

BBa_K3529012= T. reesei CBHII expression construct
BBa_K3629013= Modified P. funiculosum CBHI expression construct
BBa_K3629014= N. crassa CBHI expression construct
BBa_K3629016= Modified T. reesei EGI expression construct

To do this, the above expression constructs must be digested with BmtI and HindIII to expose the Gibson Y and Gibson X homology sequences respectively. Meanwhile, this part (BBa_K3629003) must be digested with BbsI to expose the same homology sequences. Once the digested products are placed together in the same tube with the Gibson reagents, the TEFin promoter will be swapped for the TEF1 promoter while maintaining the Lip2 signal peptide for secretion.

Gibson 1 is used to assemble this promoter with a coding sequence (with the remain 57nts of the Lip2 signal peptide at the N-terminus)into the nourseothricin destination vector (BBa_K3629015). This coding sequence must have Gibson 2 at the end to ligate into the destination vector successfully (figure 1).


Figure 1. Assembly of BBa_K3629003 with another GOI with the Lip2 signal peptide into the nourseothricin destination vector (BBa_K3629015)

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 53
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

References

1.https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-017-0819-8


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