Part:BBa_K2833018
J23104+B0032+mEos+autotranspoter+Ter
Autotranspoter refers to a family of proteins that carry sufficient information on a single gene to direct their own secretion out of a bacterial cell. ATs can insert into the OM(outer membrane) to translocate a passenger domain through the OM, so ATs can form an efficient surface display system because the native passenger domain at the N-terminal can be easily replaced by the protein of interest.[1]
We linked a fluorescent protein mEos(BBa_K2833003)to the N-terminal of autotransporter to visualize the surface display.
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 mEos cells(figure 1B, middle), while the signal was stronger in supernatant than in precipitation in mEos surface displayed cells(figure 1B, right). To further confirm this result, we removed the supernatant and observed that the signal in mEos surface displayed cells fragments was almost the same as the mEos intracellular expressed ones(figure 1C).This suggests that the surface displayed mEos were anchored to the outer membrane and being precipitated with the cell fragment, while the intracellular expressed mEos were solved in the supernatant.
Microscopy results
We observed the fluorescence under the super-resolution microscope. As figure 2 shows, The intracellular expression of mEos displayed the rode-shape of E.coli, while the signal of surface displayed mEos showed the dotted pattern around the cells, which suggested that the mEos were gathered and distributed at the surface of E.coli..
From the microscopy results, we found several interesting phenomes. One is that the longer and bigger the bacteria are, the more obviously the dotted distribution pattern around the bacteria could be seen(fig.3). One possible explanation was that the long bacteria have bigger surface area to let the displayed fluorescent protein depart from each other, while in the small bacteria, the surface displayed fluorescent protein were too crowded to show the dotted pattern. Another explanation could be that the long bacteria is more mature that the small one. In the small bacteria, the fluorescent protein were just generated but not transported to the surface yet while there is already enough time for the long bacteria to do the surface display part.
Another interesting phenome was that some autotransporter loading mEos showed the departing tendency from the outer membrane of bacteria(fig.5). Allowing for the secretion function of autotransporter, we thought that the departing-like fluorescent protein may be excreted by the autotransporter, suggesting that the system may be not very stable. However, back to our project, as long as the VHb gathered around the bacteria, it could collect oxygen to increase the local oxygen concentration then to benefit the growth of bacteria. Thus the autotransporter could be applied to our project.
Reference
[1] Edwin van Bloois, et al. Decorating microbes: surface display of proteins on Escherichia coli. Trends in Biotechnology (2011), Vol. 29, No. 2
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 872
Illegal XbaI site found at 899
Illegal PstI site found at 911 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 872
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal PstI site found at 911
Illegal NotI site found at 864
Illegal NotI site found at 929 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 872
Illegal BamHI site found at 893 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 872
Illegal XbaI site found at 899
Illegal PstI site found at 911 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 872
Illegal XbaI site found at 899
Illegal PstI site found at 911
Illegal NgoMIV site found at 794
Illegal NgoMIV site found at 1731
Illegal AgeI site found at 1506 - 1000COMPATIBLE WITH RFC[1000]
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