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Team:Berkeley/Results
From 2010.igem.org
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Results of Delivery of GFP payload with Payload Delivery Device | Results of Delivery of GFP payload with Payload Delivery Device | ||
| - | ''Experimental Set Up'' | + | '''''Experimental Set Up''''' |
In general to assay payload delivery, we first | In general to assay payload delivery, we first | ||
# Feed the payload bacteria to the choanos and induce self-lysis. Induction of self lysis and feeding were not necessarily at the same time. F | # Feed the payload bacteria to the choanos and induce self-lysis. Induction of self lysis and feeding were not necessarily at the same time. F | ||
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SInce the payload we used in our assays was GFP, we assayed for delivery events using fluorescent microscopy. We generally looked at our choanos at 63x or 100x magnification, but also used a confocal microscope. | SInce the payload we used in our assays was GFP, we assayed for delivery events using fluorescent microscopy. We generally looked at our choanos at 63x or 100x magnification, but also used a confocal microscope. | ||
| - | ''Challenges'' | + | '''''Challenges''''' |
One of the challenges we faced in assaying for payload delivery was finding a media that the choanos would be comfortable with that the payload bacteria were also able to lyse in. After assaying several different media formulations, including CMG3, LB, TB, ASW and four different mixtures of ASW and LB. What we found was that CMG3 media was the best media for choanos in which we were able to get substantial lysis. | One of the challenges we faced in assaying for payload delivery was finding a media that the choanos would be comfortable with that the payload bacteria were also able to lyse in. After assaying several different media formulations, including CMG3, LB, TB, ASW and four different mixtures of ASW and LB. What we found was that CMG3 media was the best media for choanos in which we were able to get substantial lysis. | ||
Revision as of 03:48, 26 October 2010
- 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]
Results of Delivery of GFP payload with Payload Delivery Device
Experimental Set Up In general to assay payload delivery, we first
- Feed the payload bacteria to the choanos and induce self-lysis. Induction of self lysis and feeding were not necessarily at the same time. F
- Next, we assay for successful delivery events.
SInce the payload we used in our assays was GFP, we assayed for delivery events using fluorescent microscopy. We generally looked at our choanos at 63x or 100x magnification, but also used a confocal microscope.
Challenges One of the challenges we faced in assaying for payload delivery was finding a media that the choanos would be comfortable with that the payload bacteria were also able to lyse in. After assaying several different media formulations, including CMG3, LB, TB, ASW and four different mixtures of ASW and LB. What we found was that CMG3 media was the best media for choanos in which we were able to get substantial lysis.
The major challenge we faced with assaying for payload delivery was determining the timing of when to induce self-lysis and when to look at the choanos under the microscope. For induction of self lysis, we tested timepoints from 90 minutes before feeding the choanos to 30 minutes after feeding the choanos, with 15 minute intervals. For time points before feeding, we induce self lysis in a testube, and then put it in a 37 degree shaker until it is time to feed the choanos. For time points after feeding, we add arabinose to the choanos and keep them incubating at 37 C until we assay. From our experiments, we found that induction at the same time as feeding, or 15 or 30 minutes after feeding yielded the most d
For looking at the choanos under the microscope, we tested timepoints from 1 hour after feeding to 24 hours after feeding.
From our extensive testing, we found that inducing self-lysis at at the same time as feeding, 15 minutes after feeding or 30 minutes after feeding yielded the most delivery events, and 3 to 5 hours after feeding was the best time to look at the choanos.
