Difference between revisions of "Part:BBa K1847015"
Line 8: Line 8:
 
Fusion between the ice nucleation protein (INP) and annexing V. RFP is found in the sequence to facilitate localization of plasmids.   
 
Fusion between the ice nucleation protein (INP) and annexing V. RFP is found in the sequence to facilitate localization of plasmids.   
 
Part made using the INP submitted by the Edinburg team 2011 [[Part:BBa_K523013]].
 
Part made using the INP submitted by the Edinburg team 2011 [[Part:BBa_K523013]].
−
<p>INP-Annexin V is expressed in the membrane fraction of the cell, as it is shown in Figure 1. </p>
+
===Experimental set-up===
 +
Bacteria containing INP-Annexin V in a high copy plasmid (pSB1C3) or a low copy plasmid (pSEVA371) were cultured in Lysogenic Broth (LB) with 50 &micro;g/mL (for pSB1C3) and 13 &micro;g/mL (pSEVA371) of chloramphenicol overnight at 37&ord;c with a shaking of 200 rpm. The cultures were emulsified with EmulsiFlex-C3 to obtain a cell lysate. Then, a Western blot was performed.
 +
Two different conditions were tested for each cell culture:
 +
<ul>
 +
<li>Cell lysate with no further treatment (crude).</li>
 +
<li>Cell lysate that was centrifuged for 10 min at 10,000 rpm to separate the soluble proteins from the membrane fraction (supernatant).</li>
 +
</ul>
 +
 
 +
===Results===
 +
As can be seen in Figure 1, there is expression of INP-Annexin V inside the cell and it seems that it is present on the membrane fraction.  
 
[[File:ETH15_Only_INP_annexin_gel.jpg|right|500px|thumb|Figure 1. INP-Annexin V expression.]]
 
[[File:ETH15_Only_INP_annexin_gel.jpg|right|500px|thumb|Figure 1. INP-Annexin V expression.]]
−
However, we were not able to demonstrate externalization of INP in the membrane.
+
 
  
  

Revision as of 13:10, 18 September 2015

promoter-RBS-INP-annexin V-RFP

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 2075
    Illegal NheI site found at 2098
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 475
    Illegal AgeI site found at 2684
    Illegal AgeI site found at 2796
  • 1000
    COMPATIBLE WITH RFC[1000]

Fusion between the ice nucleation protein (INP) and annexing V. RFP is found in the sequence to facilitate localization of plasmids. Part made using the INP submitted by the Edinburg team 2011 Part:BBa_K523013.

Experimental set-up

Bacteria containing INP-Annexin V in a high copy plasmid (pSB1C3) or a low copy plasmid (pSEVA371) were cultured in Lysogenic Broth (LB) with 50 µg/mL (for pSB1C3) and 13 µg/mL (pSEVA371) of chloramphenicol overnight at 37&ord;c with a shaking of 200 rpm. The cultures were emulsified with EmulsiFlex-C3 to obtain a cell lysate. Then, a Western blot was performed. Two different conditions were tested for each cell culture:

  • Cell lysate with no further treatment (crude).
  • Cell lysate that was centrifuged for 10 min at 10,000 rpm to separate the soluble proteins from the membrane fraction (supernatant).

Results

As can be seen in Figure 1, there is expression of INP-Annexin V inside the cell and it seems that it is present on the membrane fraction.

Figure 1. INP-Annexin V expression.