Part:BBa_K3034007

Surface Display System

This year, we improved a reporter device (BBa_BBa_J364000) into a surface display system (BBa_K3034007) by fusing GFP with INPNC. The improved system can anchor the downstream protein of INPNC to the surface of bacteria, while the GFP is used as the reporter gene. So, other teams who have the need of surface display can insert their target gene into this system.

Ice nucleation protein (INP) is a secretory outer membrane protein from Pseudomomas syringae, P.flurorescens and several other Gram-negative bacteria. INP can anchor one or more "passenger proteins" to the outer membrane of bacteria. The fixation of exogenous proteins on the bacteria surface through INPNC can not only greatly improve the efficiency of enzymatic reaction, but also avoid the degradation of exogenous proteins by intracellular enzymes of host cells[1].

Besides, we added a segment of linker between INPNC and GFP to ensure that two adjacent domains do not sterically interfere with one another.

Usage and Biology

Quantitative detection of fluorescence

First, we cultured the bacteria overnight and adjusted them to the same OD600. We ultrasonic broken, centrifuged and respectively resuspend precipitation to measure the distribution of GFP in E.coli DH5α carrying BBa_BBa_J364000 and E.coli DH5α carrying BBa_K3034007.

Fig.1. The relative fluorescence intensity of E.coli DH5α carrying BBa_BBa_J364000 and E.coli DH5α carrying BBa_K3034007. The relative fluorescence intensity= Fluorescence of precipitation/ (Fluorescence of supernatant+ Fluorescence of precipitation)×100%.

The results showed that both precipitation and supernatant contained relatively strong GFP. Moreover, the distribution of GFP in E.coli DH5α carrying BBa_K3034007 was not significantly different from that in E.coli DH5α carrying BBa_BBa_J364000. This was not in line with our expectation, after analysis, it may be caused by incomplete ultrasonic broken of bacteria.

Since the E.coli DH5α carrying BBa_K3034007 expressed GFP, this indirectly indicated that INPNC was successfully expressed. However, the content of GFP in the E.coli DH5α precipitate (cell membrane) carrying BBa_K3034007 was not significantly higher than the E.coli DH5α carrying BBa_BBa_J364000. We hypothesized that INPNC was expressed but the efficiency was not so high.

Microscopic observation

Next, we used fluorescence microscopy to see if the INPNC worked. E.coli DH5α carrying BBa_BBa_J364000 (GFP) was rod-shaped and the fluorescence was equably distributed in E.coli (Fig. 2a). The fluorescence of E.coli DH5α carrying BBa_K3034007 (INPNC+GFP) was observed to be dotted and dispersed on the surface of E.coli (Fig. 2b,2c). The results proved that GFP has apparently been anchored to the surface of the E.coli and INPNC was working.

In addition, we also noticed that E.coli DH5α carrying BBa_K3034007 (INPNC+GFP) had fluorescence aggregation on one side of the E.coli surface (Fig. 2c). The result is consistant with fact that we found in the literature[2] that the INPNC forms aggregates in the cell membrane.

Fig.2. The fluorescence microscopy of E.coli DH5α carrying BBa_BBa_J364000(a) and E.coli DH5α carrying BBa_K3034007 (b、c).

Conclusion

By fusing GFP with INPNC, we can implement the following improvements:

1. We upgraded this part into a surface display and report system, which can anchor GFP to the surface of E.coli and realize the function enhancement of the original part.
2. While GFP is used to report the expression of other enzymes, the system can also anchor other enzymes together with GFP to the bacterial surface to realize the surface display of certain enzymes and enhance the enzyme activity.

References

[1] Li mingya, & Lin chenshui. (2016). Ice crystal nuclear protein and its application in bacterial surface display technology. Amino acids and biological resources, 38(2), 7-11.

[2] Qiu, Y., Hudait, A., & Molinero, V. (2019). How Size and Aggregation of Ice-Binding Proteins Control Their Ice Nucleation Efficiency. Journal of the American Chemical Society, 141(18), 7439-7452.

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
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 62
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 472
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1686