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Team:Berkeley/Project/Payload
From 2010.igem.org
- Home
- Project
- Parts
- Self-Lysis
- Vesicle-Buster
- Payload
- [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2010&group=Berkeley Parts Submitted]
- Results
- Judging
- Clotho
- Human Practices
- Team Resources
- Who We Are
- Notebooks:
- [http://www.openwetware.org/wiki/Berk2010-Daniela Daniela's Notebook]
- [http://www.openwetware.org/wiki/Berk2010-Christoph Christoph's Notebook]
- [http://www.openwetware.org/wiki/Berk2010-Amy Amy's Notebook]
- [http://www.openwetware.org/wiki/Berk2010-Tahoura Tahoura's Notebook]
- [http://www.openwetware.org/wiki/Berk2010-Conor Conor's Notebook]
Overview
"Payload" refers to the contents of a bacteria that are desired to be delivered to the cytoplasm of the Choanoflagellate. Payload can come in many forms: proteins, DNA, etc.
Green Fluorescent Protein Payload
In our Payload Delivery assay, we used green fluorescent protein (GFP) as our protein payload because it's delivery can be easily detected through use of fluorescent microscopy. To fill bacteria with our GFP payload, we transformed them with a plasmid containing our construct (INSERT NAME), which is GFP under a constitutive promoter. Then we turned them into delivery machines by transforming them again with our payload delivery device.
To see the results of this assay, visit our results page.
Targeted Fluorescent Protein Payload
In order to further confirm delivery, we delivered GFP fused to a nuclear localization tag {NLS) along with normal RFP. We hoped to observe Choanoflagellates that were fluorescent red throughout and had glowing green nuclei. However, we only saw red and green throughout, meaning the NLS wasn't functional in Choanoflagellates.
Our next step is going to be trying different nuclear localization tags used in other eukaryotes as well as constructing our own based off of BLASTs of the Choanoflagellate's genome for NLS-like sequences.
Transposase Payload
Now that we have confirmed successfully protein deliver, the next step is to test more interesting protein payloads, in particular, proteins that could make Choanoflagellates genetically tractable such as transposases. Over the Summer, we constructed a large portfolio of transposase parts and constructs.
Future Payloads
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.
