IGEM:IMPERIAL/Protocols/I13208

Part I13208: AiiA Enzyme Test

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 990μL 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 1μL IPTG
• To the +ZN ependorfs add 1μL Zinc Chlroide
• Add 10μL of 500nM AHL to all tubes

Not Finished