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From 2010.igem.org

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Our project consists of 3 devices: Self-lysis, Vesicle Buster, and Payload. Read below for more information on each of the devices.

Team:Berkeley/Project/Self_Lysis Team:Berkeley/Project/Vesicle_Buster Team:Berkeley/Project/Payload Team:Berkeley/Results

Self-lysis Device

To break through the E. coli’s inner and outer cell wall, we used the self-lysis device derived from the 2008 UC Berkeley iGEM team (registry name and picture of 2008 part). There were, however, several challenges we needed to overcome before the device could be applied in a choanoflagellate. First, the lysis device had to be inducible by an exogenous molecule. For practical reasons we needed to be able to control when self-lysis occurred and for biosafety reasons self-lysis could not occur outside of a laboratory culture. For example, we initially constructed the self-lysis under a magnesium based promoter but that construct was not used because magnesium is commonly found in sea water and mammalian cells and would lead to lysis in undesirable and potentially dangerous situations. By putting the device under the control of an arabinose-induced promoter, Pbad, we were able to induce lysis only when desired and prevent incidental lysis. Moreover, self-lysis had to be fast acting: the bacteria had to lyse itself after it was ingested by the choanoflagellate but before it was digested by the choanoflagellate. The 2008 part took closer to five hours to lyse but we estimated that choanoflagellates digestion takes only one to two hours; therefore we needed a faster acting device. To accomplish this we added BRB to the original construct. BRB degrades the inner cell membrane. With this addition, we were able to have lysis occur within an hour after induction.

Although the self-lysis device must be under control of an exogenous inducer, self lysis must also occur in the choanoflagellates culture. In the in vitro assay for self-lysis, E. coli were grown and induced in TB media. However, choanoflagellates cannot survive in TB or LB media. Similarly, an in vitro assay showed that self-lysis failed to occur when the bacteria were put in the artificial sea water used to culture choanoflagellates. We found a compromise between the health of choanoflagellates and the activity of the self-lysis device by using Choano Growth Media 3 (CGM3). As shown in the graph below, where a decrease in optical density indicates successful lysis, the bacteria were able to lyse themselves in CMG3 media almost as well as they did in TB.

Vesicle Buster

We derived the vesicle buster device from a construct built in the Anderson Lab and intended to be used in a mammalian system. There were several design challenges the vesicle buster had to satisfy in order to properly function in our delivery scheme. Because of the short time window between ingestion and digestion, the vesicle buster had to be constitutively expressed and ready to act upon self lysis. Stable expression of the vesicle buster was accomplished by placing it under the control of Pcon, a constitutive promoter. Since the bacteria stably express the vesicle buster, the device also cannot harm the bacteria and must act only on the choanoflagellate’s membrane. This specificity was satisfied by using PFO and PLC. PFO acts only on a cholesterol-based membrane and does not affect E. coli’s cell wall. PLC also targets phsopholipids found only in eukaryotic membranes. Finally, once the food vesicle is opened and its contents are released into the cytoplasm, PLC and PFO must be prevented from breaking down any other membrane and creating further damage to the choanoflagellate. For this reason, degradation tags were added to these enzymes.

Payload

Our payload delivery device consisted of the self-lysis device and the vesicle buster device put together in one plasmid. We made competent strains of E. coli that expressed the payload delivery device and to complete delivery, we had only to create a payload plasmid to transform into the payload-delivery competent cells. By separating the payload and the payload delivery device into two different plasmids, our design made it easy to construct different payloads.

Our first payload was GFP. We hypothesized that in the case of successful delivery, we would go from observing GFP contained within the the food vesicle to GFP diffused throughout the entire cytoplasm of the choanoflagellate. As you can see in the phase pictures overlapped with fluorescent images, we were able to deliver GFP to the cytoplasm of the choanoflagellate. We also used a confocal microscope to take z-stacks of the same choanoflagellates and to demonstrate that the GFP was truly in the cytoplasm.

The next step is to target our payload to the nucleus to deliver the machinery necessary to genetically modify these organisms. Because choanoflagellates have not been well characterized, we have yet to find a functional NLS tag. Once we find a nuclear localization signal we will be able to deliver the zinc finger and transposase payloads that we have already constructed. The zinc fingers can be used to knockout genes and the transposases can be used to knock in genes. In addition we have constructed payload plasmids that are not meant to be integrated into the genome but are intended to be expressed extrachromosomally. A simple way to detect whether we establish stable expression of exogenous genes is to deliver DNA that codes for GFP and run the choanoflagellates through a flow cytometer.