Difference between revisions of "Part:BBa K1603000"
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<p>The fluorescent microscopy pictures from the detection test with CEN.PK2 containing construct 4 (C4) are shown in figure 3-6. Samples were made with different amounts of concentrated P-factor and compared with wild type CEN.PK2 (WT).</p>
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<p>The fluorescent microscopy pictures from the detection test with CEN.PK2 containing construct 4 (C4) are shown in figure 1-5. Samples were made with different amounts of concentrated P-factor and compared with wild type CEN.PK2 (WT).</p>
 
<p>[[File:ChalmersGothenburgPostitiveControl.jpg|350px]]
 
<p>[[File:ChalmersGothenburgPostitiveControl.jpg|350px]]
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<br><b>Figure 3. Positive control. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.</b></p>
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<br><b>Figure 1. Positive control. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.</b></p>
 
<p>[[File:ChalmersGothenburgC4230.jpg |350px]]
 
<p>[[File:ChalmersGothenburgC4230.jpg |350px]]
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<br><b>Figure 4. C4 with 230 µl concentrated P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.</b></p>
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<br><b>Figure 2. C4 with 230 µl concentrated P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.</b></p>
 
<p>[[File:ChalmersGothenburgC40.jpg|350px]]
 
<p>[[File:ChalmersGothenburgC40.jpg|350px]]
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<br><b>Figure 5. C4 without P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.</b></p>
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<br><b>Figure 3. C4 without P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.</b></p>
 
<p>[[File:ChalmersGothenburgWT230.jpg|350px]]
 
<p>[[File:ChalmersGothenburgWT230.jpg|350px]]
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<br><b>Figure 6. WT with 230 µl concentrated P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.</b></p>
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<br><b>Figure 4. WT with 230 µl concentrated P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.</b></p>
 
<p>[[File:ChalmersGothenburgWT0.jpg|350px]]
 
<p>[[File:ChalmersGothenburgWT0.jpg|350px]]
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<br><b>Figure 7. WT without P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.</b></p>
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<br><b>Figure 5. WT without P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.</b></p>
 
<p>Only the C4 samples containing the highest amount of P-factor showed a few fluorescent cells, which is promising for our concept. The weak signal can be explained by the fact that constructs containing the amplification system through dCas9-vp64 could not be completed. This forces the detection system to rely on the weak promoter pFUS1 <b>[1]</b> to express mRFP. This can result in a weak fluorescent signal when the P-factor is detected.</p>
 
<p>Only the C4 samples containing the highest amount of P-factor showed a few fluorescent cells, which is promising for our concept. The weak signal can be explained by the fact that constructs containing the amplification system through dCas9-vp64 could not be completed. This forces the detection system to rely on the weak promoter pFUS1 <b>[1]</b> to express mRFP. This can result in a weak fluorescent signal when the P-factor is detected.</p>

Revision as of 21:28, 18 September 2015

Fusion GPCR STE2MAM2

Fusion GPCR of the non-cytoplasmic N-terminal signal peptide from STE2 (Saccharomyces cerevisiae) and Pheromone P-factor receptor MAM2 (Schizosaccharomyces pombe) without its signaling peptide. Allows in vivo detection of the Pheromone P-factor from S.pombe through the Pheromone pathway in S.cerevisiae. 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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 408
  • 1000
    COMPATIBLE WITH RFC[1000]


To evaluate the detection properties of this part, STE2MAM2 was connected to the promoter pTDH3

The fluorescent microscopy pictures from the detection test with CEN.PK2 containing construct 4 (C4) are shown in figure 1-5. Samples were made with different amounts of concentrated P-factor and compared with wild type CEN.PK2 (WT).

ChalmersGothenburgPostitiveControl.jpg
Figure 1. Positive control. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

ChalmersGothenburgC4230.jpg
Figure 2. C4 with 230 µl concentrated P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

ChalmersGothenburgC40.jpg
Figure 3. C4 without P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

ChalmersGothenburgWT230.jpg
Figure 4. WT with 230 µl concentrated P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

ChalmersGothenburgWT0.jpg
Figure 5. WT without P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

Only the C4 samples containing the highest amount of P-factor showed a few fluorescent cells, which is promising for our concept. The weak signal can be explained by the fact that constructs containing the amplification system through dCas9-vp64 could not be completed. This forces the detection system to rely on the weak promoter pFUS1 [1] to express mRFP. This can result in a weak fluorescent signal when the P-factor is detected.