Difference between revisions of "Part:BBa K4743006"
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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.
 
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.
  
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<html><img style="width:700px" src="https://static.igem.wiki/teams/4743/wiki/folder-for-parts/pnuc-nadv-prsa-length.png" </html>
  
 
<center>Figure 2. PCR check. </center>
 
<center>Figure 2. PCR check. </center>
  
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<html><img style="width:700px" src="https://static.igem.wiki/teams/4743/wiki/folder-for-parts/pm-associated-sequence-test.png" </html>
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<center>Figure 3. The GFP flourence of the membrane.</center>
 
<center>Figure 3. The GFP flourence of the membrane.</center>

Revision as of 10:14, 11 October 2023


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]