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From 2011.igem.org

--Monday 8/15/11--

We got our sequences from last week in and checked them to make sure they looked good. Not all of the parts were correct, but we are about a day or two from being done with construction! Here is a table of which parts are completed and which parts we need to complete in order to finish construction.

	LacI	c1	TetR
Repressor Screening	*R0010+E0240+I13453+B0034	R0051+E0240+I13453+B0034	*R0040+E0240+I13453+B0034
Promoter Screening	E0240+I13453+B0034+C0012	E0240+I13453+B0034+C0051	E0240+I13453+B0034+C0040
Wild Type Controls	*R0010+E0240+I13453+B0034+C0012	R0051+E0240+I13453+B0034+C0051	*R0040E0240+I13453+B0034+C0040

'	LacI	c1	TetR
Repressor Screening	R0010+E0240+I13453+B0034	R0051+E0240+I13453+B0034	R0040+E0240+I13453+B0034
Promoter Screening	E0240+I13453+B0034+C0012	E0240+I13453+B0034+C0051	E0240+I13453+B0034+C0040
Wild Type Controls	R0010+E0240+I13453+B0034+C0012	R0051+E0240+I13453+B0034+C0051	R0040E0240+I13453+B0034+C0040

In order to finish our construction, we are going to cut out R0010 and R0040 as inserts and put them in front of the parts needed. Tomorrow We will transform and hopefully have the finished construct by Wednesday.

--Tuesday 8/16/11--

Ligated and transformed yesterday's digestion.

--Wednesday 8/17/11--

Once again, their are quite a few colonies on our vector control plates. Fortunately, our ligation involved putting promoters (R0010 and R0040) in front of parts containing GFP (E0240+repressor). This means that a successful ligation/transformation will produce a green colony. There were no green colonies on our vector controls which indicated that any green colonies were successful ligations! We did a quick PCR screen and confirmed that this was the case. We now have the nine parts we need to screen and characterize our mutants!

--Thursday 8/18/11--

Yesterday we cultured our four completed construction so that we could miniprep them today. The plan now is to cut our three repressor screening constructions (Promoter+E0240+I13453+B0034) at Spe1 and Pst1. This will allow us to ligate these parts with our repressor mutants (cut at Xba1 and Pst1). The digestion of our construct looked very odd on a gel. We decided to set up the digestion again.

--Friday 8/19/11--

Once again, the digest looks very odd when run on a gel. We either see a weird gradient of bands instead of solid bands, or solid bands that are higher than the expected number of bases. We are suspicious of our enzymes, and have decided to set up a digest that will test their efficiency. We digested a random part 4 times with only one restriction enzyme in each digestion. Running this along side the uncut plasmid should tell us how well the enzyme is cutting. We got mixed results, but overall can conclude that our enzymes are working. We set up the digestion again and will extract it from a gel on Monday.