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Team:Berkeley/Project/Payload
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| + | 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. | ||
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| + | 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. | ||
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| + | 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. | ||
Revision as of 01:58, 25 October 2010
- Home
- Project
- Parts
- Self-Lysis
- Vesicle-Buster
- Payload
- Parts Submitted
- Results
- Judging
- Clotho
- Human Practices
- Team Resources
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.
