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From 2010.igem.org

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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.

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.