"

From 2014.igem.org

Picking colonies

Picked colonies from 200uL plated PUC19 transformed cells, 10uL plated pBbe1a (- ctrl) transformed cells, and 10uL pBbe1a CsgA Arc’ed cells. There were many colonies for the PUC19 and pBbe1a (- ctrl) that were good for picking, however the Arc’ed transformation plates had better colonies for picking than the heat shock transformations for the pBbe1a CsgA plasmid. We obtained a plate of LSR10 pBbe1a CsgA plated cells from Bom. We picked 2 colonies from each plate and inoculated them in 3mL of YESCA + Carb for Congo Red staining. They were left shaking at 37C for 3 hours. After 3 hours, liquid YESCA cultures were induced with IPTG and transferred to 25C shaking incubator to grow up overnight.

Mini Prep

The same 2 colonies picked for Congo Red experiments were picked from Turbo pBbE1a-CsgA Arc’ed transformed cells and from the Turbo pBbe1a (- ctrl) cells and were inoculated in 5mL of LB+Carb and left shaking at 37C for 5 hours. We picked 1 culture from each to miniprep and nanodrop. Stored at -20C

Congo Red Spot Test

In 96-well plate:

A1 LSR10-1 A2 LSR10-2 A3 pBbe1a(- ctrl)-1 A4 pBbe1a(- ctrl)-2 A5 pBbe1a-CsgA-1 A6 pBbe1a-CsgA-2 A7 PUC19-1 A8 PUC19-2

Spotted on YESCA+CR+CBB+IPTG+Carb plates (x3 for practice)

Starting cultures for LSR10 electrocompetent cells

Picked 4 colonies from Bom’s LSR10 plate and inoculated in 5mL pure LB. Left in 37C shaking overnight

We also began to work on our wiki. We looked at some of the winning wikis from years past and discussed their layouts and design decisions.