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Revision as of 03:12, 4 October 2012

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.

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!