Team:Groningen/Notebook/6 July 2009

From 2009.igem.org

Wet

Transformation of E. coli TOP10 with BBa_J23109, BBa_J23100 and BBa_J23106

• BBa_J23109 iGEM plate 2, 2G
• BBa_J23100 iGEM plate 1, 18C
• BBa_J23106 iGEM plate 1, 18O

All dry plasmid DNA in the wells was dissolved in 15ul sterile diH2O, and transfered to 500ul cups.

• Four cups with 50ul competent E.coli TOP10 cells were thawed on ice, and 2ul of dissolved plasmid was added.
• After 30 min. on ice, the cups were put in a heat block at 37C for 5 min. and followed by 5 min. on ice.
• To the cells, 800ul TY medium was added and incubated at 37C for 1 hour.
• 100ul was plated on TY-Agar-Amp plates and incubated o.n. at 37C.
• The remainer was centrifuged for 1 min. at full speed and the cells were resuspended in 100ul TY-medium, and plated on TY-Agar-Amp plated. The plates were stored at 37C o.n.

Inoculation of TY-medium for Glycerol Stocks

• GVP cluster vector BBa_J61035 (2x) in E.coli TOP10
• Terminator vector ? (2x) in E.coli TOP10

• 4.5 ml TY-medium with Amp. was inoculated with a colony grown on TY-Agar-Amp. plates o.n.
• The tubes were put in a waterbath at 37C (150 rpm) and grown o.n.

Purification of HmtA (PA Q9I147 6xHis Tag)

Preculture

• inoculaton of a colony in 5 ml LB+amp
• Incubate overnight 37°C with shaking

Dry

We found that the dimensions given in Walsby1994 are in fact outer dimensions, instead of inner dimensions as we first assumed. Furthermore mass/density calculations were added to our computational model of the geometry of gas vesicles.

It was found to be problematic to determine the buoyant density of E. coli with gas vesicles, as the volume of the bacteria can differ quite a bit (both from strain to strain and during the life cycle) and we also haven't found very concrete data about the number of gas vesicles produced (nor any direct measurements of the buoyant density of E. coli with gas vesicles). So instead we've decided to give a lower bound on the (volume) fraction of the cell that should consist of gas vesicles.