Part:BBa_K4743006

PnuC-GFP-Pm associated sequence

Design

PnuC codes for the transporter that transport NR into the cell and transport NMN out of the cell. We endeavored to express pnuC on the cell membrane of kluyveromyces marxianus, a close relative of Saccharomyces cerevisiae, to pump NMN out. However, pnuC we use is derived from Bacillus mycoides. Hence, to express pnuC on the cell membrane of kluyveromyces marxianus requires a signal peptide called plasma membrane associated (PMassc) sequence. PMassc sequence is derived from the last fifty amino acids of Gap1 transporter of Saccharomyces cerevisiae, guiding Gap1 transporter to the cell membrane of Saccharomyces cerevisiae. In our project, we fuse PnuC, GFP, and PMassc sequences together, so PnuC can be expressed on the plasma membrane of Kluyveromyces marxianus and we are able to observe it.

Figure 1. The map of construct.

Test

Then, we integrated this construct into the pklac2 expression vector and integrated the plasmid into the genome of Kluyveromyces maraxianus. To test the effectiveness of PMassc sequence on pnuC, we put the engineered k. marxianus under the fluorescence microscope and observe fluorescence on the cell membrane of K. marxianus, indicating the PMassc sequence can make proteins be expressed on cell membrane of kluyveromyces marxianus.

Figure 2. PCR check.

Figure 3. The GFP flourence of the membrane.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1761
  Illegal XbaI site found at 484
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1761
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1761
  Illegal BglII site found at 88
  Illegal BamHI site found at 16
  Illegal XhoI site found at 682
  Illegal XhoI site found at 1408
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1761
  Illegal XbaI site found at 484
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1761
  Illegal XbaI site found at 484
  Illegal AgeI site found at 1774
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Black, W.B., Aspacio, D., Bever, D. et al. Metabolic engineering of Escherichia coli for optimized biosynthesis of nicotinamide mononucleotide, a noncanonical redox cofactor. Microb Cell Fact 19, 150 (2020). https://doi.org/10.1186/s12934-020-01415-z

2. Popov-Čeleketić, D., Bianchi, F., Ruiz, S. J., Meutiawati, F., & Poolman, B. (2016). A Plasma Membrane Association Module in Yeast Amino Acid Transporters. The Journal of biological chemistry, 291(31), 16024–16037. https://doi.org/10.1074/jbc.M115.706770