Team:Berkeley/Project/Payload

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<font size=5>'''Green Fluorescent Protein Payload'''</font>
<font size=5>'''Green Fluorescent Protein Payload'''</font>
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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.  
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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.  
To see the results of this assay, visit our results page.  
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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 a wide varitey of 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.
 
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.
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 07:04, 26 October 2010

Payload header.png


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

Transposase Payload

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.

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