Team:Penn/SurfaceDisplay

We sought to create a system in which iGEM teams and labs can display any protein of their choosing on the surface of E. coli. We a novel Suface Display BioBrick (BBa K811004) using the N and C terminal domains of the Ice Nucleation Protein (INPNC) which allows iGEM teams to fuse any desired protein to INPNC using a simple ligation protocol with BamHI and PstI restriction sites.

As a preliminary proof-of-concept for our INPNC-enabled system, we displayed the red fluorescent protein mCherry on the outer membrane of E. coli BL21. After sonication and centrifugation of induced cells, almost all of the mCherry was localized in the membrane fraction when fused to INPNC, whereas in the control Intein-mCherry fusion (which exhibits cytoplasmic localization), all of the mCherry was contained in the lysate (Figure 1).

Figure 1

Figure 1: Surface Display of mCherry using INPNC system. INPNC-mCherry and Intein-mCherry fusions were expressed in E. coli BL21 in the pET26b expression vector and Wood-Intein expression plasmid, respectively. When fused to INPNC, almost all mCherry was localized in the membrane fraction after sonication and centrifugation, while in the case of Intein-mCherry, all mCherry was localized in the cytoplasmic lysate.

Having demonstrated that our INPNC surface display system could localize a desired protein (mCherry) to the outer membrane surface, we sought to display the picomolar affinity, HER2 binding Designed Ankyrin Repeat Protein (DARPin) H10-2-G3 on the surface of our BL21 cells. This would allow for bacterial targeting to HER2 over-expressing cells, which could then be lysed in a spatially accurate manner using our light-activated ClyA expression system. Such surface display of a large antibody-mimetic protein is unprecedented, and assaying whether it had actually occurred would be difficult.

We constructed a C-terminal INPNC-DARPin fusion in the same manner as our INPNC-mCherry fusion, except we added a Human Influenza Aggregation (HA) tag to the N-terminal of DARPin, to allow us to use antibodies to assay whether it had been localized to the membrane. After expression and 48-hour induction of INPNC-DARPin-HA in BL21 cells using the pET26b expression vector, we immunostained cells using an anti-HA, Alexa-fluor 647 conjugated antibody (Cell Signaling Technologies). Since antibodies are not permeable to the E. coli membrane, after washing the cells there should only be signal on those cells which have displayed the HA tag on their surface. We attempted to assay these stained cells with flow cytometry, but soon realized that we required much higher-resolution FACS sorting equipment than we had available to count bacteria.

We then took a small amount of HA immunostained bacteria and visualized them by confocal microscopy at high (630x) magnification using a 633 He-Ne laser. Using this immunostaining visualization method, we were able to demonstrate for the first time that DARPin H10-2-G3 had in fact been localized to the cell surface (Figures 2-9)!

We first stained control bacteria expressing DARPin-HA without fusion to INPNC (Figures 2-3). The following images were taken of identical densities of bacteria as verified by phase contrast.

Figure 2

Figure 2: Uninduced pET26b-DARPin-HA (-IPTG) bacteria are not immunostained by anti-HA antibody.

Figure 3

Figure 3: Induced pET26b-DARPin-HA (+IPTG) bacteria are not immunostained by anti-HA antibody.

Then, to determine whether INPNC could display an HA tag on the surface of E. coli, we stained bacteria expressing INPNC-HA and found that IPTG-induced bacteria exhibited strong red fluorescence when excited by the 633nm laser (Figures 4-5).

Figure 4

Figure 4: Uninduced pET26b-INPNC-HA (-IPTG) bacteria are not immunostained by anti-HA antibody.

Figure 5

Figure 5: Induced pET26b-INPNC-HA (+IPTG) bacteria are strongly immunostained by anti-HA antibody, indicating surface expression.

Finally, to determine whether we could display our HA-tagged DARPin on the surface of E. coli, we stained INPNC-DARPin-HA expressing bacteria and found strong immunofluorescence under the induced condition (Figures 6-7).

Figure 6

Figure 6: Uninduced pET26b-INPNC-DARPin-HA (-IPTG) bacteria are not immunostained by anti-HA antibody

Figure 7

Figure 7: Induced pET26b-INPNC-DARPin-HA (+IPTG) bacteria are strongly immunostained by anti-HA antibody, indicating surface expression.

We also repeated the experiment without the antibody to rule out any possibility of autofluorescence. As expected, no fluorescence was detected when INPNC-DARPin-HA bacteria were not stained with the anti-HA Alexa-Fluor 647 antibody (Figures 8-9).

Figure 8

Figure 8: Uninduced pET26b-INPNC-DARPin-HA (-IPTG) (-AB) bacteria are not fluorescent.

Figure 9

Figure 9: Induced pET26b-INPNC-DARPin-HA (-IPTG) (-AB) bacteria are not fluorescent.

All controls performed as expected, and we are very confident that our DARPin has been displayed on the surface of E. coli.