Team:Berkeley/Parts

From 2010.igem.org

Our project consists of 3 devices:
Self-lysis
Vesicle Buster
Payload

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). However, there were 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 placed 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. 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 must lyse itself after it is ingested by the choanoflagellate but before it is digested by the choanoflagellate. The 2008 part took closer to five hours to lyse but we estimated that choanoflagellates digest over the course of two to three 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 inducing with arabinose.

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. An in vitro assay of self lysis conducted in the artificial sea water that choanoflagellates are cultured in proved to be unsuccessful. We found a compromise between the health of choanoflagellates and the activity of the self-lysis device by using CMG3 media. 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 designed 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 switch between different payloads.