"
Team:Berkeley/Results
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]
Results of Delivery of GFP payload with Payload Delivery Device
General Experimental Set Up
In general to assay payload delivery, we first
- Feed the payload bacteria to the choanos and induce self-lysis with arabinose. Induction of self lysis and feeding were not necessarily at the same time.
- 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.
Challenges
One of the challenges we faced in assaying for payload delivery was finding a media that the choanos liked and that the payload bacteria were able to lyse in. After assaying several different media formulations, including CMG3, LB, TB, ASW and four different mixtures of ASW and LB, 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. F or looking at the choanos under the microscope, we tested timepoints from 1 hour after feeding to 24 hours after feeding.
From our experiments, we found that induction at the same time as feeding, or 15 or 30 minutes after feeding yielded the most delivery events. For timepoints before 15 minute before feeding, the number of delivery events drops off fairly steeply. SUPPORT WITH HEMOCYTOMETER DATA
We also observed that choanos seem to eat and digest bacteria with 1-2 hours after feeding, and that the best time to look for successful delivery was between 3 and 5 hours after feeding.
Assay Results and Controls
Specific Protocols or 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.
