Team:UESTC-China/Improve

description

We improved this reporter device into a surface presentation + reporting system (BBa_K3034007) by fusing GFP with INPNC so that the team could make reporter genes through GFP and anchor the target protein to the bacteria outer membrane for more applications.

Ice nucleation protein (INP) is a secretory outer membrane protein from Pseudomomas syringae P. flurorescens and several other Gram—negative bacteria[1]. INP can anchor one or more "passenger proteins" to the outer membrane of E.coli DH5α. The fixation of exogenous proteins on the cell 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.

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

Quantitative detection of fluorescence

We first cultured the bacteria overnight and made OD600 uniform. we ultrasonic broken, centrifuged and respectively resuspend precipitation to measure the distribution of GFP in E.coli DH5α carrying BBa_J364000 and E.coli DH5α carrying BBa_K3034007.

Fig.1. 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 after centrifugation.Moreover, the distribution of GFP in E.coli DH5α with BBa_K3034007 was not significantly different from that in E.coli DH5α with BBa_J364000. But, the content of GFP in the broken E.coli DH5α with BBa_K3034007 was higher than that in the E.coli DH5α with BBa_J364000.

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 that in the control group (with BBa_J364000). We hypothesized that INPNC was expressed but not highly active.

Microscopic observation

Next, E.coli DH5α with BBa_J364000 (GFP) was observed to be rod-shaped and fluorescently filled with E.coli DH5α under a 40-fold microscope.The fluorescence of E.coli DH5α with BBa_K3034007 (INPNC+GFP) was observed to be dotted and dispersed on the surface of E.coli DH5α. The results proved that INPNC was successfully expressed and functioned.

Fig.2. The fluorescence microscopy of E.coli DH5α carrying BBa_J364000(a) and E.coli DH5α carrying BBa_K3034007 (b、c).

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

Results

The part we improved was successfully expressed and worked, but INPNC was inefficient.

Improvement and application

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] Yang, X., Sun, S., Wang, H., & Hang, H. (2013). Comparison of autotransporter and ice nucleation protein as carrier proteins for antibody display on the cell surface of Escherichia coli. Prog Biochem Biophys, 40, 1209-19.
[2] Lee, S. Y., Choi, J. H., & Xu, Z. (2003). Microbial cell-surface display. Trends in biotechnology, 21(1), 45-52.