IGEM:MIT/2006/Notebook/2006-7-14

TO DO

Andre: re-do SPP PCR (followed by PCR cleanup & gel); find out about exponential phase promoter (make liquid culture for miniprep if possible), research other smells

Stephen: Site-directed mutagenesis of ATF1 + gel; Make indole knockout cells competent (to be followed by transforming Dudareva plasmid); re-do SPP PCR (followed by PCR cleanup & gel), make liquid culture of coding region + terminator transformants, make RBS cultures

Veena: GC on different BSMT concentrations and indole knockout, order primers for BAT2, THI3, pCHBA

Indole Knockout Strain

The indole knockout strain was made competent using the following protocol:

http://openwetware.org/wiki/Preparing_chemically_competent_cells

Site-directed mutagenesis for ATF1

Redone using the Strategene manual available here: http://openwetware.org/wiki/Endy:Site-directed_Mutagenesis

We got the primers ready for mutagenesis using the following protocol:

http://openwetware.org/wiki/PNK_Treatment_of_DNA_Ends

Liquid cultures

We got many, many transformants hopefully carrying SAMT connected to B0015 and BSMT connected to B0015. We made 4 liquid cultures from each type of colony. We also made two liquid cultures from the glycerol stock of cells containing the ribosome binding sites B0030 and B0032. I plan to come in tomorrow to miniprep each of these 10 liquid cultures for eventual sequencing (in the case of the SAMT.B0015 and BSMT.B0015 cultures) and ligation (for each culture).