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Guelph/19 June 2008
From 2008.igem.org
Dear Team,
These are the minutes for the Technical Presentation by Dave on Thursday.
Upcoming: 2008 June 24 11:00am to end of day - Laboratory learning, discussion and primer design. 2008 June 23 12:00pm Noon - Meeting at the Grad Lounge.
Present: Dave Johnston, Brendan Hussey, Eddie Ma
Regrets: Lisa Ledger, Jen Vo
Dave presented us with some appendices to the iGem binder. The appendices are in regards to transformation procedures (not electroporation) that the competition officials have tested and know to work with the biobricks-- we'll likely continue with our present electroporation techniques since we're familiar with them and since it has worked for us so far. The appendices also refer to promoter testing which tests the concentration of a product specified by a gene to see how well the promoter is responding to a molecule that activates transcription for that gene. We have not definitively decided whether or not to follow that protocol yet since we're heading away from the biobrick strategy on the present step, developing our own operons for now.
Dave has struck a deal with New England Biolabs (NEB); we'll be receiving enzymes from them at a 50% discount. Dr. Allen-Vercoe has provided us with the Bifido, E. coli and Lactobacilli strains. Dr. Manish has signed on our behalf a Materials Transfer Agreement (MTA) on behalf of the team for the broad host range plasmid pDSK-GFPuv, so we shall be expecting the full sequence to be sent to us. Dave and Dr. Manish have drawn up a budget. Recombinant TAQ in E. coli and JM109 arrived; we'll need to work with this. Brendan, Lisa and Tim have figured out their cloning strategy.
The broad host range plasmid should work with gram negative and gram positive bacteria, though we'll do the actual transformation in order to find out for sure-- this is the reason for the broad host range plasmid.
We will likely use Dr. Emma-Vercoe's gastrointestinal simulating machine to test the growth of any gastrointestinal bacteria we transform.
The next steps are very clear, we will design primers for the vitamin A enzyme genes and the phenylalanine genes.
Dave describes the RNAi project. Interfering RNA [RNAi] is double stranded RNA. This RNA is the complimentary sequence for the transcription of a gene coming from some genome sequence. In plants, animals, it has been demonstrated that RNAi can be used to knock out gene transcription products because overhangs from the double stranded RNA are replicated by cellular machinery. The replication products are carried by cellular machinery that search for endogenous complimentary mRNA and break them apart; furthermore the RNA machinery flood the cell and bind to the DNA responsible for the target RNA's original sequence and so the DNA becomes bound to the flooding RNA and unable to transcribe. The proceeding experiment is a proof of concept that demonstrates that RNAi as produced from a bacteria which is designed to complement some gene transcribed by a plant is able to perform the same knocking out of gene translation product. The gene that Dave is interested in is called TB1, which is responsible for down regulating the branching behaviour in modern corn-- modern mature corn usually has one long stalk and doesn't branch. If we introduce bacteria that produce RNAi against TB1, then the resulting plant will appear more like an ancestor of corn which branches a lot instead.
Please see attached, Dave's presentation.
See particularly Dave's slide about Techniques and Workflow-- please note that Ed will be doing a _strict_ search for the iGem guidelines tomorrow while in the lab with Brendan and Lisa-- you are all welcome to attend as there will be some lab work introduction and primer design as well-- please see other e-mail for details.
Thanks,
