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From 2012.igem.org

For outreach we wanted to make colourful or glowing E. Coli.

Registry Part	Plasmid	Well	Plate	Promoter	Characteristic(s)	Gene
BBa_I13521	pSB1A3	6O	2	TetR	repressible	Red Fluorescent Protein (RFP)
BBa_I13522	pSB1A2	8A	2	TetR	repressible	Green Fluorescent Protein (GFP)
BBa_I13600	pSB1A2	24E	1	TetR	repressible	Cyan Fluorescent Protein (CFP)
BBa_I712052	pSB1AK8	13G	2	T7	bacteriophage	Luciferase
BBa_K274210	pSB1A2	6N	3	TetR	repressible	Orange Fluorescent Protein (OFP)

We chose five different genes from the IGEM parts registry that were fluorescent or bio-luminescent (as shown in the table above): red, blue, orange and green fluorescent proteins, as well as luciferase. The fluorescent proteins were all transformed into Top 10 competent E-coli as they have TetR repressible promoters, while Luciferase it was transformed into BL21-DE3 (Origami) competent E. coli cells as it has a T7 promoter. All competent cells were obtained from Robert Willows lab.

The Top 10 E. coli cells were divided into 4 x 50 μl aliquots, while 100 μl of BL21 was measured out. We located the corresponding wells for the gene plasmids in the plates supplied in our parts kit, and resuspended each plasmid in 10 μl of MilliQ H20. We then added 1μl of each plasmid to the relevant E.coli cells for transformation. We incubated the cells for 5 mins on ice, subjected them to heat shock at 42°C, and placed them on ice for a further 2 mins. We then added 500 μl of SOC media to each tube and incubated them for an hour at 37°C in a shaker.

1mL of 0.1M IPTG stock solution was added to 9mL of MilliQ H2O giving a final concentration of 0.01M. This final IPTG was then used for the plates.

Each of the transformations were then plated out on ampicillin plates as follows: 1 x 20 μl on plates with added IPTG
1 x 200 μl on plates with added IPTG
1 x 20 μl on plates without IPTG
1 x 200 μl on plates without IPTG

The plates were then incubated overnight at 37°C.

• Due to using 20 ampicillin plates from our stock of plates for use with our project, we made up 20 more LB agar plates on this day as well.