Construction

From 2006.igem.org

September 3, 2006

Now that we knew the enzymes are working, we proceeded to test the MiniPreps from 2005 and 2006 (O = match, X = no match):

We also began the transformation of some of the 2005 parts:

• I0500 (Promoter inducible by pBad/araC – part of thermometer)
• C0056 (DNA sequence for repressor protein 434 cI)
• I12006 (Modified lamda Prm promoter repressed by 434 cI)
• E0422 (RBS.ECFP.Terminator)

September 1, 2006

    With a lot (a LOT) of help from Seema, we now have 24 Eppendorf tubes of competent cells (DH5a-z1) in the -80C freezer.  We also have ~10 plates with Ampicillin (Amp), ~8 plates with Kanamycin (Kan), and 4 plates with Amp/Kan resistance.  Seema also sorted out our iGEM box and separated between the 2005 parts and the 2006 parts as well as grouped the enzymes, reagents, and ladders.

Due to a lot of problems with previous attempts at transformations and quantifications, we decided to begin testing the enzymes as well all the part lengths so that we can get a handle of what we have to work with. So we tested all the enzymes, (EcoRI, XbaI, SpeI, and PstI) using a test plasmid. In theory, the uncut plasmid should move farther in the gel than the cut plasmid because the circular plasmid is more compact and would better be able to move through the agarose gel. This was indeed the case and all the enzymes cut successfully as shown the by the 4 bands being at the same height above the uncut band.

One thing to note was that the enzyme Xba1 worked slower than the others. Since the idea was to just check if the enzymes were working, we didn’t need to let the enzymes cut for the full 1 hr (as per the protocol). Thus, we ran the gel about 40 min early and saw two bands (one for each of the cut and uncut plasmid) in the Xba1 lane.

August, 11, 2006

Hey team,

It looks like we'll soon be underway and get to start building and testing parts. I seem to remember Farshid saying he and Jessica already extracted the parts that we needed from the registry, so that we can move on from there. There's a semi-final outline of what we'd like to do on the Critical Design Review (CDR) page so take a look at that. Once we get approval from Prof. Davies, we can get started.

The overall idea is to "assign" a part to a member so that member is responsible for growing/maintaining that part as well as adding that part to someone else's part. Also, depending on other people's thoughts on this, it would be a good idea to double up on the parts so that we get at least two cracks at making a part at the same time. This allows everyone to participate in the process from start to finish. Of course there will be a lot of flexibility in who does what, but this gives us some sort of baseline to start with. I'll be making some sort of sign-up sheet so if you want to make a specific part for some reason you can. I was thinking of using Google calendar to help with the coordination, so if you want a Gmail address, you can just email me and I can invite you (Gmail is actually really good). I'll keep you updated on this idea though.

Finally, CHECK THE WIKI REGULARLY!! We are now going to start doing stuff so there should be regular postings on the Wiki and so I don't want to flood everyone's inboxes. I will try to see if there is some way to indicate whether a link has been modified in some way so that you know there has been an update. Or if any of you know how to implement that by all means go for it.

Charles Yoon

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