Part:BBa_K3629011

Nourseothricin resistance gene

Nourseothricin acetyltransferase (nat1) gene from Streptomyces noursei.

Usage and Biology

This is the coding sequence for nourseothricin resistance, which is a robust selection marker that can be used in many eukaryotes, specifically yeast, including Yarrowia lipolytica -the chassis for our project. Particularly for Y. lipolytica there are not many available auxotrophy-based selection markers due to the limited number of auxotrophic strains publically available for use. Therefore, antibiotic resistance genes are a suitable alternative. To use this selection marker, 50-200µg/mL of nourseothricin is added to media depending on the organism.

The part is a key part of our collection and functions as a destination vector for other Y. lipolytica parts when cloned into a plasmid with a promoter and terminator sequence attached.

Design

This coding sequence was attached to the TEF promoter (BBa_K2117000), and the XPR2 terminator (BBa_K3629004) in creation of the expression construct for this part (BBa_K3629015). We provided the fully functional expression construct in our collection for teams who want to transform and use this part in our modular Gibson Assembly system designed to integrate recombinant DNA directly into the Y. lipolytica genome. However, just the coding sequence is provided here in case teams want to use this coding sequence for different purposes.

Specifically in our collection, which provides expression constructs for cellulases and amino acid synthesis genes, BBa_K3629015 can be assembled with another one of those genes, creating a larger gene cassette. The larger cassette can then be linearized with NotI and transformed into <Y. lipolytica </i>

The expression constructs in our collection that can be assembled with the nourseothricin expression construct are:

• BBa_K3629012= 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
• BBa_K3629017= T. reesei EGII expression construct
• BBa_K3629018= N. patriciarum BGS expression construct

Sequence and Features

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]

Codon optimized for expression and function in Yarrowia lipolytica.