Part:BBa_K1847015
promoter-RBS-INP-annexin V-RFP
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE 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 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 475
Illegal AgeI site found at 2684
Illegal AgeI site found at 2796 - 1000COMPATIBLE 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 12.5 µg/mL (pSEVA371) of chloramphenicol overnight at 37⩝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.
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