IGEM:IMPERIAL/Protocols/I13208

Motivation
The motivation behind testing this to part is to test the Zinc Hypothesis, and to test qualitatively if AiiA is hydrolysing AHL.

Materials & Equipment

 * Equipment
 * Wallac Victor 3 Multi-Well Fluorimeter
 * Ependorf Tubes
 * Gilson Pippettes
 * 37oC Shaker


 * Materials
 * AHL
 * GFP Standard Solution
 * E.coli Growth Medium w/Ampicilin (LB/M9)
 * E.coli DH5a Culture Containing I13208

Protocol

 * Inoculate two cultures each from 10ul of stored I13208 in 2ml of both LB and M9 growth medium containing 50ug/ml Ampicilin.
 * Incubate at 37oC for overnight in a shaker.
 * Following day, prewarm LB/M9 to 37oC by placing in the 37oC incubator
 * Measure and record OD600_1 in report sheet
 * Inoculate a 2ml fresh culture from the o/n to bring back the OD600 to 0.1, use prewarmed LB/M9 + Ampicilin.
 * Volume used to inoculate new culture = (0.1/OD600_1)*2ml
 * Return LB/M9 to incubator
 * Incubate new cultures at 37oC for 2 hours in a shaker -  This returns cells to exponential phase 
 * Measure and record the OD600_2 in report sheet
 * Dilute again for an OD of 0.1 in a new culture of 10ml of a prewarmed LB/M9 + Ampicilin.
 * Volume used to inoculate new culture = (0.1/OD600_2)*10ml
 * Vortex new I13208 cultures.
 * Pipette 990 of each of the two cultures into 4 labelled ependorf tubes
 * M9/+IPTG/+ZN
 * M9/+IPTG/-ZN
 * M9/-IPTG/+ZN
 * M9/-IPTG/-ZN
 * LB/+IPTG/+ZN
 * LB/+IPTG/-ZN
 * LB/-IPTG/+ZN
 * LB/-IPTG/-ZN
 * To the +IPTG ependorfs add 1 IPTG
 * To the +ZN ependorfs add 1 Zinc Chlroide
 * Add 10 of 500nM AHL to all tubes

Not Finished