Revision as of 06:52, 11 October 2018

J23104+B0032+INP+sfGFP

Ice nucleation protein(INP)is a secretory outer membrane protein from Pseudomomas syringae P. flurorescens and several other Gram—negative bacteria.In the bacterial surface display technology，INP is widely used as a functional carrier protein.

Design

We got the INP from BBa_K523013 constructed by iGEM11_Edinburgh. To test the surface display efficiency of INP, we added sfGFP to the downstream of INP, and expressed the fusion protein under the control of J23104 and B0032.

Cell lysis result

We conducted a cell lysis experiment to observe the distribution of fluorescent protein(figure 1). From this result we can see that after cell lysis followed by centrifuge, both the precipitation and supernatant shows relative strong fluorescence signal in intracellular expressing GFP cells(figure 1B, middle), while the signal was stronger in supernatant than in precipitation in surface displaying cells(figure 1B, right). This suggests that the surface displayed GFP were anchored to the outer membrane and being precipitated with the cell fragment, while the intracellular expressed GFP were solved in the precipitation.

Fig.1 the fluorescence of intracellular expression of GFP and surface display of GFP via INP before and after cell lysis. A: before cell lysis; Right: after cell lysis

Microscopy result

We observed the fluorescence under the super-resolution microscope. As figure 2 shows, The intracellular expression of GFP displayed the rode-shape of E.coli, while the signal of surface displayed GFP showed the dotted pattern, which suggested that the GFP were gathered and distributed at the surface of E.coli..

Fig.2 super-resolution microscopy of INP-sfGFP Left: intracellular expression of GFP; Right: surface display of GFP via INP

Sequence and Features