IGEM:IMPERIAL/2006/Protocols/J37015

Part J37015: Prey Cell Positive Feedback Characterisation

Motivation

The motivation behind this test construct is to assess the length and amplitude of the exponential phase. This is essential to comply with the Lokta-Volterra constraints.

Equipment & Materials

• Equipment
  • Wallac Victor 3 Multi-Well Fluorimeter
  • Ependorf Tubes
  • Gilson Pippettes
  • 37[[:Category:{{{1}}}|{{{1}}}]] Shaker
  • Microfuge

• Materials
  • E.coli Growth Medium w/Ampicilin
  • E.coli Culture Containing T9002
  • E.coli Culture Containing J37015
  • GFP Standard Solution

Protocol

Preparing Cultures for testing

• Inoculate a culture from 10μL of stored J37015 into 2ml LB/M9 growth medium containing 50μg/ml Ampicilin.
• Also innoculate a culture of T9002 from 10μL of stored T9002 into 2ml LB/M9 growth medium containing 50μg/ml Ampicillin.
• Incubate at 37[[:Category:{{{1}}}|{{{1}}}]] for overnight in a shaker.
• Following day, measure and record the optical density for both cultures at 600nm in a report sheet.
• Dilute in order to achieve an optical density of 0.1. To do this, inoculate Xml Overnight Culture into Yml Fresh Culture LB/M9 + Ampicilin (prewarmed using the 37[[:Category:{{{1}}}|{{{1}}}]] incubator- avoids temperature shock) using the formula below. Total volume will be 8ml.-OD600= 0.1 is the optical density (at 600 nm) of an arbitrary dilution of bacteria, used in order to standardise our measurements whilst testing the prey cell positive feedback loop. This optical density is dilute enough to facilitate the entry of the bacteria into exponential growth phase again (they reach stationary phase after being grown overnight)
  • Volume of Overnight Culture = (0.1/OD600_1)*8ml
  • Volume of fresh Culture = 8-(Overnight Culture)

• Incubate at 37[[:Category:{{{1}}}|{{{1}}}]] for 2 hours in a shaker - This returns cells to exponential phase
• Measure and record the OD600_2 of both cultures.
  

• Dilute T9002 again for an OD of 0.1 (using formula below) in a new culture of 15ml of a prewarmed LB/M9 + Ampicilin (You do not need to dilute J37015 at this stage).
  • Volume of Overnight Culture = (0.1/OD600_2)*15ml
  • Volume of Fresh Culture = 1 - Overnight Culture

• The cultures are now ready to be tested

• Assessing the positive feedback loop base state:
  • Take 4x 200μL samples from each J37015 culture and pipette into a 96 well plate
  • Add 200μL of 200x diluted GFP control to a well.
  • Add 200μL of LB/M9 to a well
  • Use the Victor3 to measure GFP output and absorbance - This is to assess the state of the positived feedback loop DA- We expect this to be completely switched off presumably?
• To the J37015 cultures add Theophylline for a final concentration of 10mM for J37015RS or xxxxx of IPTG for J37015Cre DA- Need to find out what concentration of IPTG is!!! Low concentration should be sufficient.
• Taking samples to assess AHL levels:
  • Take 1ml of J37015 cell culture into an eppendorf
  • Spin down cells for 20 seconds
  • Pipette supernatent from J37015 into newly labelled eppendorf tube
  • Take samples every 10 minutes
  • After a period of 60 minutes stop taking samples
• Assessing AHL levels
  • Take 1ml samples of T9002 into eppendorfs
  • Spin for 20 seconds JS: Does it need to be 20 sec? Can we do it for 5 sec only...?
  • Discard the supernatent
  • Resuspend T9002 cells in J37015 supernatent
  • Pippete 4x 200 μL samples into a 96 well plate
  • Incubate plate in shaker at 37[[:Category:{{{1}}}|{{{1}}}]]
  • Wait for 4 hours for GFP to reach steady state
  • Measure and record OD and Flourescece

Potential Issues

• Cells have to spun down, which makes them a little unhappy
• Unsure of what level of repression we'll get from the various control mechanisms
• Unsure of the speed of the positive feedback loop

comments from Vincent

• Depending on the strengh of the positive feedback, it might be interesting to explore different dilutions to begin with.