We are in Week 4 of the iGEM project.
The debrief session was attended by Professor Freemont. A number of issues were raised; these are described below.
The work done on the various projects was reported at the debriefing session.
- General protocols available for E Coli experimentation were recorded.
- Work was started on protocols specific to the Biofilm project
- It was reported that 4 constructs would be employed. Also, it was suggested, that due to some parallels with last year's Imperial project, these established constructs could already be exploited.
- More work needs to be done in terms of reporter mechanisms, e.g. DSRed.
- This project proceeds by employing "test" constructs until the required HRP constructs are made available.
- Can E Coli Polymerase be employed in the in-vitro/in-veso system, or is T7 the only possibility?
- DSRed, was suggested as a possible reporter system; especially since this would already be characterized from one of the other projects. Are there other reasons which could/could not warrant its use in the Biofilm project? - especially, consider time-scale of operation.
- The constructs to be pursued further were discussed.
- Relevant protocols to be developed per device.
- Email Christina from the "Vesicles" Division concerning considerations/issues/problems raised including:
- Stability/Rigidity of vesicles
- No. of ribosomes possibly accommodated by vesicles - in other words, size considerations.
- Polymerase restrictions - only T7 employable?
- Methods of fabrication
- Materials employed - particular w.r.t. phospholipid compostion/mixture used.
- Storage of vesicles, especially at temperatures of 4 degrees Celsius.
- Communication methods across membrane.
- Alternative means of forming pores within membrane, since a-hemolysin toxin is not permitted during our project work.
- Also, included in this email, should be the relevant research papers on which our methods/project work to date has been based.
- Proposed new reporter mechanism, involving DSRed - inherent long degradation time.
- Issues overlapping with In-Vitro/In-Veso systems, e.g. non-T7 polymerase, etc.
- Cmv was initially proposed as a constitutive promoter; however, this can only be used successfully within mammalian cells; not prokaryotic cells.
- The list of experiments to be conducted should be completed.
- (other issues covered? input required)
Ambitions by end of week
- Start production of media within lab.
- Hopefully start cloning for E Coli System.
- Organize division of labour between different projects, and, within the projects themselves, especially w.r.t labwork.