IGEM:MIT/2006/Notebook/2006-8-10

to do

1. GC work with new smell samples -rescheduled for 8/11
2. Miniprep all LCs/make appropriate glycerols/sequence -done
   • Remember to Make 10 biobrick named glycerols and send for sequencing -done
3. Make LCs of IK transformants in pm (overnight smell test and regular batch for glycerols/am smell test) -done
4. PCR pchBA with 5% Betane -done
5. site-directed mutagenesis on ATF1 in banana generating device (with both RBS30 and RBS32), THI3 in TOPO vector with NEW mut primers -done

PCR of pchBA

...it worked! The following showed bands on the gel and are currently in the fridge (pcr cleaned up):

• pchBA pcr'ed w/ new forward and reverse primers, with 2μL betaine added to pcr rxn [53.3 ng/μL]
• pchBA pcr'ed w/ new forward and reverse primers, with 4μL betaine added to pcr rxn [9.9 ng/μL]
• pchBA pcr'ed w/ new forward and reverse primers, with a 96deg (instead of 94deg) denature step...we stopped the reaction on cycle 21 out of 30 b/c we needed the block for mutagenesis [17.8 ng/μL]
• pchA pcr'ed w/ new reverse primer (and the old forward one), with 2 μL betaine added to pcr rxn [64.2 ng/μL]

next steps: biobrick, do the pchA mutagenesis, hook it up to terminator, RBS, promoter!

Indole KO Strain

The following table summarizes the results of the indole KO transformations.

	# of Transformants	Notes about colonies
pUC18	lots	essentially uniform in size (though denser areas contain smaller colonies); well spread across plate
J45100	~110	many tended to grow in clusters; range of sizes from very small to large (though large colonies may be more than one)
J45120	~70	not as many clusters; the colonies are huge, in general (a few small ones, though)
J45170	~100	not as many clusters; the colonies are huge, in general (a few small ones, though)
J45200	~110	range of sizes from very small to large; some clusters
J45220	~50	a few small colonies but most are huge; few clusters

Mutagenesis

1. Held off on BAT2 because of Spe1 site
2. Regrew THI3 culture because not enough cells
3. Mutagenesis on 4 ATF1 (200,220,250,270)
4. Did PNK step http://openwetware.org/wiki/PNK_Treatment_of_DNA_Ends and mutagenesis protocl from http://openwetware.org/wiki/Knight:Site-directed_mutagenesis