This year, we improved this reporter device (BBa_J364000) (Fig. 1a) into a surface display system (BBa_K3034007) (Fig. 1b) by fusing GFP with INPNC (BBa_K523008). 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.
Fig. 1. Schematic map of BBa_J364000 (a) and BBa_K3034007 (b).
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. In our experiments, without linker, GFP could not be properly expressed.
Quantitative detection of fluorescence
First, we cultured the bacteria overnight and adjusted them to the same OD600. We ultrasonic broke, centrifuged and respectively resuspend precipitation to measure the fluorescence intensity of GFP in E.coli DH5α carrying BBa_J364000 and E.coli DH5α carrying BBa_K3034007 (Fig.2).
Fig. 2. The relative fluorescence intensity of E.coli DH5α carrying 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_J364000. There are some differences with our expectation, after analysis, we think that 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_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_J364000 (GFP) was rod-shaped and the fluorescence was equably distributed in E.coli (Fig. 3a). 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. 3b,3c). The results proved that GFP has apparently been anchored to the surface of the E.coli and INPNC worked.
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. 3c). The result is consistant with the fact that we found in the literature[2] that the INPNC forms aggregates in the cell membrane. Thus, we are more confident that we have achieved our improvement.
Fig. 3. The fluorescence microscopy of E.coli DH5α carrying BBa_J364000(a) and E.coli DH5α carrying BBa_K3034007 (b、c).
1.We improved BBa_J364000. GFP is an intracellular reporter gene. And we achieved the expression of GFP on the surface of E.coli.
2.Based on the current results, there is space for further improvement in the efficiency of system expression (The activity of INPNC needs to be improved).
3.Other teams who have the need of surface display can insert their target gene into this system. For example, the system can be applied to whole-cell biocatalysts, heavy metal contamination adsorption, antigen and antibody display, etc.
[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.
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