Part:BBa_K5351002

NFS1mu

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 800
  Illegal BglII site found at 997
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Origin

Synthesized by the team and constructed as a mutant of the NFS1 gene in *Saccharomyces cerevisiae*.

Properties

BBa_K5351002 (NFS1mu) is a mutant of NFS1 where the Ile residue at position 492 of NFS1 is mutated to Asn. This mutation is used to verify whether the mutation at this position will enhance the xylose metabolism of the strain.

Usage and Biology

NFS1mu is significant in biofuel production as it supports research into improving yeast strains' ability to metabolize xylose. The mutation at position 492 was specifically designed for this purpose.

Experimental Approach

We constructed pSCm-NFS1mu by ligating the pSCm backbone and the target gene NFS1. The pSCm was digested to obtain a linear backbone with a length of 5984 bp.

Figure 1. Gene map of NFS1.

Fig. 2 indicates the band is consistent with the results.

Figure 2. Gel electrophoresis validation of the linear pSCM-N20 plasmid backbone.

These backbones were ligated with the target gene NFS1 and transformed into competent *E. coli* DH5α. Fig. 3a shows the presence of single colonies on the plate. Colony PCR was performed to validate the colonies.

Figure 3. Monoclonal antibody validation and sequencing of pSCm-NFS1 (DH5α) (a: pSCm-NFS1 colonies; b: Gel electrophoresis validation of colony PCR of pSCm-NFS1; c: Sequencing result of pSCm-NFS1).

Colony PCR results (**Fig. 3b**) showed bands of 288 bp consistent with the expected length, thus validating our successful plasmid construction. The colonies were also sent for sequencing. According to the results shown in **Fig. 3c**, the NFS1 gene was successfully ligated to the backbone without any apparent mutations, confirming the successful construction of the pSCm-NFS1 plasmid.

Reference

Wei F., Li M., Wang M., et al. (2020). A C6/C5 co‐fermenting *Saccharomyces cerevisiae* strain with the alleviation of antagonism between xylose utilization and robustness. *GCB Bioenergy*, 13(1), 83–97.

Brat D., Boles E., Wiedemann B. (2009). Functional expression of a bacterial xylose isomerase in *Saccharomyces cerevisiae*. *Applied Microbiology and Biotechnology*, 75, 2304-2311.