Team:MIT bexp


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bacterial experimental protocol

In order to image cells transformed with our improved toggle and composite parts, we adhered to the following protocol.

Experimental Protocol

M9 Minimal Top Agar.
To 733.9 mL autoclaved deionized H2O, add 200 mL of 5x M9 minimal salts (56.4g Bacto M9 minimal salts, 5x from Difco in 1L H2O, autoclaved to sterilize), 10mL 40% glycerol (80mL glycerol in 120mL H2O, autoclaved to sterilize), 2mL 1M MgSO4 (24.65g MgSO4*7H2O in 100mL H2O, autoclaved to sterilize), 100uL CaCl2 (14.7g CaCl*2H2O in 100mL H2O, autoclaved to sterilize), 20mL 10% Casamino acids (50g Bacto Casamino acids from Difco in 500 mL H2O, autoclaved to sterilize), and 32 mL 10 mg/mL thiamine (10 mg per mL H2O, use 0.22 um filter to filter-sterilize).

Prepare cell lawns for UV exposure.
    Escherichia coli type JM2.300 transformed with either pTSMa or pLPTa and a composite biobrick (one of K415006, K415013, K415022 or K415023) were grown 12-14 hours in 4mL aliquots of LB at 37°C with Kanamycin and Ampicillin, and IPTG to a final concentration 2.65 uM. An OD measurement was taken to confirm the cells to be in the log phase of growth. The remainder of the protocol was performed in the dark. The IPTG was washed out in two cycles of pelleting, decanting, and resuspending the pellet in the original volume of fresh LB. The cells were then concentrated 10x. This sample was added 1:40 to 4mL melted M9 minimal top agar and kept at 50°C while Kanamycin and Ampicillin were added. If the composite biobrick was not K415022 or K415023, the mixture received M Acyl Homoserine Lactones (AHL) to a concentration of 0.3uM. The mixture was vortexed, quickly poured into small petri dishes, and allowed to cool for 5 minutes. The plates then grew at 30°C for 4 hours.

Expose cell lawns to UV.
    After 4 hours of growth, the E.coli lawns received varying powers of UV exposure (from 1 to 100 uJ/mm^2) through a mask designed to expose some cells to and hide others from the light. The cells then grew, agar up, at 30°C for 4 hours. (details on setup...)

Mask cutting.

Image cells with Zeiss.