MIT Synthetic Biology Working Group Standardized FACS Protocol Draft 1 – 9/20/04

This protocol sets out to standardize the components of a FACS experiment necessary to allow repeatable and comparable results. The output of these experiments is typically fluorescence from a genetic regulatory network.

Contents 1 Media and Glassware 2 Strains and Plasmids 3 Protocol 4 Analysis and Instrument settings

Media and Glassware

Provisionally, supplemented M9 media will be used for all cultures. The media recipe used by CC and JCB is –

• 1x M9 Minimal Salts (from Sigma):
• 12.8 g/L NaHPO4.7H20
• 3.0 g/L KH2PO4
• 0.5 g/L NaCl
• 1.0 g/L NH4Cl
• 0.1mM MgSO4
• 2mM CaCl2
• 0.4% Glucose/Glycerol
• 0.1% Casamino Acids

Cultures should, where possible, be grown in flasks rather than tubes. To allow sufficient aeration, flasks should be able to hold five times the culture volume and tubes should hold 10 times the culture volume. Evaluation of the difference in using a flask versus a tube would be a simple and worthwhile experiment.

Strains and Plasmids

It is unrealistic to specify one strain and plasmid for all characterization work but initial discussion suggests that, where possible, MC4100 and a 3-series plasmid should be used. Consequently, these will be used for the standard positive and negative control. The current positive control (assembled by RPS) is – As E0430 is the output of many of our experiments, it may make sense to change the positive control to I6031, this is the same as the current positive control but replaces B0014 with B0015. The negative control should be MC4100 containing an empty 3-series plasmid.

Protocol

1. Inoculate 5ml of supplemented M9 minimal media from a streaked plate. The o/n should be grown in a tube.
2. Grow the culture for 12-14 hours on a roller at 37oC.
3. Perform a 1/1000 dilution in minimal M9 media. This culture should be grown in a flask. If possible, record the OD600 prior to, and immediately post, dilution to allow possible refinement of the protocol.
4. Record OD600 at each time point. Samples can be left on ice for up to 6 hours without affecting results significantly. Further discussions should consider setting a standard OD600 or time post dilution at which measurements should be taken to reduce the need for long time course experiments.
5. If early time points will be taken, the 1/1000 dilution should be carried out using pre-warmed media.

Analysis and Instrument settings

Trials with the positive and negative controls will allow the determination of an appropriate calibration of the fluorescence PMTs to make the controls suitable for a range of constructs and plasmids.

Further discussion should also specify a standard method for gating and data analysis.