Difference between revisions of "Part:BBa K4743006"
Line 3: Line 3:
 
<partinfo>BBa_K4743006 short</partinfo>
 
<partinfo>BBa_K4743006 short</partinfo>
  
−
PnuC codes for the transporter that trnsport NR into the cell and transport NMN out of the cell. We also integrate this part with GFP and Plasma membrane associated sequence. To check if this transporter is on the plasma membrane.
+
<span class='h3bb'><big>'''Design'''</big></span>
  
−
<span class='h3bb'><big>'''Overall metabolic pathway of NMN'''</big></span>
+
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.
  
−
PnuC is involved in the pathway of Astaxanthin production in PTSH-Taiwan's project.
 
  
−
<html><img style="width:800px" src="https://static.igem.wiki/teams/4743/wiki/folder-for-parts/nmn-metabolic-pathway.png" </html>
+
<span class='h3bb'><big>'''Test'''</big></span>
  
−
<center>Figure 1. The Matabolic pathway of NMN production.</center>
+
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.
  
−
<span class='h3bb'><big>'''function of parts'''</big></span>
 
−
 
−
Intend to check the PnuC is on the membrane we've check the green light of the membrane
 
−
 
−
<html><img style="width:400px" src="https://static.igem.wiki/teams/4743/wiki/folder-for-parts/pm-associated-sequence-test.png" </html>
 
−
 
−
<center>Figure 2. The GFP flourence of the membrane .</center>
 
−
 
−
<span class='h3bb'><big>'''Length checking of the sequence'''</big></span>
 
−
 
−
<html><img style="width:800px" src="https://static.igem.wiki/teams/4743/wiki/folder-for-parts/pnuc-nadv-prsa-length.png" </html>
 
−
 
−
<center>Figure 3. The legnth of this sequence .</center>
 
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Revision as of 10:07, 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.


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.

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]