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From 2012.igem.org

pDawn Characterization

To characterize our pDawn gene expression system, we showed the following:

1. pDawn allows for light-dependent gene expression in bacteria
2. pDawn allows for light-dependent lysis of mammalian cells by bacteria

Light Dependent Gene Expression in Bacteria

We tested for light dependent gene expression by cloning in an mCherry reporter protein into the multiple cloning site of the pDawn system. First, we tested for the on-off ratio by growing cultures of BL21-pDawn-mCherry in both inducing and non-inducing conditions for 22 hours. After spinning down the cultures in a centrifuge, we were able to visually confirm the expression of mCherry due to the bacterial pellet grown in inducing conditions to be colored red, while the other pellet had no color (Figure 1)

Figure 1

We then characterized the induction kinetics of the pDawn system through an mCherry expression time course. We induced cultures of BL21 pDawn-mCherry for 0 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, or 22 hours in a 37C incubator shaking at 225 rpm, and then transferred them into a dark incubator under the same condition for the remaining growth period. After 24 hours, mCherry fluorescence was read on a Tecan Infinite m200 plate reader and normalized by OD. The cultures were then spun down in a centrifuge. These results can be seen in Figure 2.

Figure 2

After verifying that the pDawn system was able to express mCherry in a light-dependent manner, we substituted our ClyA for the mCherry gene to prove that ClyA could be expressed in a light dependent manner. We grew cultures of BL21 pDawn-His-ClyA in both inducing and non-inducing conditions, lysed the cells, and purified the ClyA with His affinity Ni beads. We ran the purified protein on a 4-12% Bis-Tris 1mm SDS-PAGE gel stained with Invitrogen SimplyBlue gel stain, along with various controls. These results can be seen in Figure 3. As shown, pDawn was able to express ClyA in a light-dependent manner.

Figure 3

Light-Dependent Lysis of Mammalian Cells by Bacteria

We then wanted to prove that our pDawn-ClyA construct was able to lyse mammalian cells in a light-dependent manner. To assess this, we plated BL21 bacteria transformed with pDawn-ClyA or pDawn-mCherry on Columbia Agar plates supplemented with 5% Sheep Blood (BD). These plates are used to qualitatively detect hemolytic activity in bacteria by visually confirming lysis through a color change in the media as the blood cells are lysed. After plating the bacteria, cultures were grown in non-inducing conditions at 37C until visible colonies were present (~12 hours). Plates were then grown at 25C under either inducing or non-inducing conditions for 24 hours and imaged. These results are visible in Figure 2.

Figure 4

pDawn and Nissle 1917

In order to further develop our system for future in vivo therapeutic applications, we transformed Nissle 1917 with pDawn-mCherry to see if we could implement our system into a non-pathogenic strain of E. coli. We repeated our initial experiments and achieved light-dependent gene expression in Nissle 1917 for the first time ever. We are now hoping to clone in our pDawn-ClyA construct to show that Nissle 1917 is capable of light-dependent lysis of mammalian cells. Stay tuned!

Figure 5

Generalized Surface Display System

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.

Displaying an Engineered Cancer Cell Binder on the Outer Membrane Surface

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

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

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

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

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.

Testing the Displayed HER2 Darpin on Cancer Cells

After verifying that the DARPin was displayed on the cell surface, we sought to test whether it could allow binding to HER2-overexpressing cancer cells. We were generously provided SKBR3 cells by the lab of Matthew Lazzara. These cells are derived from breast cancer cells and over-express HER2. We also obtained Human Embryonic Kidney (HEK) 293T cells, which express a low amount of HER2, to act as controls.

To verify that our SKBR3 cells overexpressed HER2, we immunostained for HER2 (primary anti-Neu Mouse Igg, Santa Cruz Biotechnology and secondary donkey anti-mouse Alexa-Fluor 647 conjugate, Jackson Immunoresearch) and visualized the cells on a confocal microscope. As expected, HER2 was overexpressed in SKBR3 cells (Figure 10).

Figure 10: SKBR3 cells over-express HER2 on their surface. Red indicates HER2 (Alexa-Fluor 647), Blue indicates DAPI.

To assay whether our DARPin-displaying E. coli bound to SKBR3 cells preferentially, we conducted experiments in which our bacteria were co-incubated with SKBR3 or HEK293T cells. The DARPin-displaying bacteria were co-transformed with eGFP for easy visualization. These experiments are still in progress to optimize the co-incubation and gentamycin protection protocol (we designed this type of experiment ourselves since it has not been done before). However, preliminary results are exciting and show preferential binding to SKBR3 cells (Figures 11 and 12).

Figure 11: HEK293T cells do not express a large amount of HER2, and DARPin-displaying bacteria do not preferentially bind to them. Red indicates HER2 (Alexa-Fluor 647), Blue indicates DAPI. Green indicates eGFP. Bacteria were co-incubated at a Multiplicity of Infection of 200:1 and washed three times after co-incubation.

Figure 12: SKBR3 cells overexpress HER2 and DARPin-displaying bacteria bind to their surface. Red indicates HER2 (Alexa-Fluor 647), Blue indicates DAPI. Green indicates eGFP. Bacteria were co-incubated at a Multiplicity of Infection of 200:1 and washed three times after co-incubation.

In summary, we demonstrated surface display of DARPin H10-2-G3 for the first time and have preliminary evidence showing our DARPin-displaying bacteria can target cancer cells. We also developed a generalized surface display BioBrick for any iGEM team to use.