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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, fed these bacteria to Choanos, and induced lysis.
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, untagged RFP. We hoped to observe Choanoflagellates that had a fluorescent red cytoplasm and had glowing green nuclei. However, we saw red and green throughout, meaning the NLS wasn't functional in Choanoflagellates.
Our future plans include trying other nuclear localization tags that have proven successful in other eukaryotes as well as constructing our own based off of BLASTs of the Choanoflagellate's genome for NLS-like sequences.
Plasmid Payload
Our delivery scheme also has the potential to deliver DNA. We currently are in the process of testing the delivery of several plasmids to the Choanoflagellates, some made by the King Lab and others by the Anderson lab. Most of the plasmids are pQXIN vectors with both SV40 and colE1 origins, containing eGFP under an animal promoter, such as Atub, Ef1a, and Myo. If the plasmids can be successfully delivered, replicated and expressed in the Choanoflagellates, they should glow green, allowing use to detect successful hits by using Flow Cytometry. This wasn't the case with our GFP delivery scheme, because there would be no way to distinguish Choanos that just ate fluorescent bacteria from successful delivery events. However, flow cytometry can be employed for Plasmid Payload, because the bacteria don't express the GFP on the plasmid they're delivering.
Transposase Payload
We're excited to take this project to the next level by delivering proteins that could make Choanoflagellates genetically tractable. Over the Summer, we constructed a large portfolio of transposase parts and constructs. Transposases are enzymes capable of randomly inserting sequences of DNA flanked by short terminal repeat sequences into other sequences of DNA.
Our constructs contain the following three transposases:
- Tn5
- Sleeping Beauty
- PiggyBac
