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Assignments Due

• Journal Club Presentation (see schedule in OWL-Space Resources)

Overview of Experiment

YOU WILL HAVE TO COME IN THE DAY BEFORE LAB DAY 7 AND PULL COLONIES:
BBa_I13522 (pTetR-GFP): pick FOUR colonies per team and grow overnight in 3 ml LB-Amp (50 µg/ml) at 37°C with shaking (250 rpm)

1. With your Sharpie, circle well-isolated colonies on the plates
2. Put 3 ml of LB + antibiotic in sterile plastic tubes (label as needed directly on the tubes)
3. Gently swab a circled colony with a sterile pipet tip or toothpick--do not touch any other colonies
4. Eject the tip into the appropriately labeled tube
5. Slightly loosen the lids and put the tubes in the 37°C shaker overnight
6. Store the plates at 4°C.

Electroporation of Mutated Plasmids

1. Prechill two 1 mm electroporation cuvettes and microcentrifuge tubes on ice
2. Thaw the electroporation-competent cells (NEB 5-alpha Electrocompetent E. coli) on ice (~10 min.) and mix by gently flicking
   
   You must be extremely gentle when working with competent cells. These cells are highly sensitive to temperature changes and/or mechanical lysis. Mix cells by gently tapping the tube or swirling with a pipet tip, not by pipetting up & down or vortexing.
   
   
3. Transfer 45 µl of cells to a chilled microcentrifuge tube
4. Add 1 µl of PCR reaction to cells and gently mix
5. Place the chilled cuvette on its side and carefully transfer cells + DNA without introducing bubbles
6. Gently tap the cuvette until the mixture of cells and DNA settles evenly to the bottom (i.e., there is no gap across the cuvette)
7. Wipe outside of cuvette with KimWipe and slide the cuvette into the electroporation chamber until the cuvette connects with the electrical contacts
8. Pulse sample ONCE at 1.7 kV, 200 ohms, 25 µF (Bio-Rad Electroporator); typical time constant = 4.8-5.1 milliseconds
   
   **Record the time constant (in ms) and the actual volts (kV) delivered to sample**
   time constant (τ): the amount of time required for the actual voltage of the delivered pulse to decay to 1/e (37%) of the initial voltage {τ = Resistance (R) x Capacitance (C)}
   
   
9. QUICKLY remove the cuvette from the chamber and add 950 µl of prewarmed (40-44°C) sterile SOC medium to the cells
10. With a sterile Pasteur pipet, quickly but gently resuspend the cells and transfer the cell suspension to a 17 mm x 100 mm round-bottom culture tube
11. Incubate the sample at 37°C for 1 hour with shaking at 250 rpm
12. Pipet 200 µl of transformed cells on a LB-ampicillin plate
13. Pour ~10 sterile solid glass beads onto the plate, set the plate on the benchtop, and "shake" plate in a perpendicular motion; invert plate to pour off beads (collect in a large beaker -- these can be reused)
14. Pellet the cells for 20 sec at 5,000xg, remove the supernatant, and resuspend in 200 µl SOC
15. On a second LB-amp plate, pipet 200 µl of resuspended cells and REPEAT step 13.
16. Let the plates sit 5 minutes at room temperature so that the liquid absorbs into the agar
17. Incubate the plates (upside down) overnight at 37°C

Fluorescent plate reader training (Keck 201)

A. Fluorescence of purified GFP

Make 10-fold and 100-fold, dilutions of the stock GFP in PBS.  In a 96-well plate, prepare an array of dilutions of purified GFP:

• plate 100 µl of each dilution in triplicate
• plate 100 µl PBS in triplicate as a control

B. Functional analysis of simple circuit: BBa_I13522 (pTetR-GFP)

Experimental goal: Does the genetic circuit work?

• Outline of goals: How do you get a big signal?
  • How do you know that what you're measuring is actually what you want?
  • What are the appropriate controls?
  • How do we account for background?
  • How does signal noise affect the results?
• Fluorescence signal detection
  • wash vs. unwashed cells
  • determine density of cultures
  • determine fluorescence output

Prepare cells: cells + R0040 (promoter only); cells + I13522 (GFP)
1. In a 1.5 ml tube, pellet 500 µl of each culture at 5,000xg for 20 sec
2. Carefully remove supernatant and resuspend pellet in 500 µl PBS or 25% glycerol
3. Prepare a 1:10 sample in a second 1.5 ml tube by adding 100 µl resuspended cells to 900 µl PBS or glycerol
4. Plate 100 µl of resuspended cells in triplicate in a 96-well plate (one set for each culture and one set for each 1:10 culture)
5. CONTROLS: plate 100 µl of unwashed cells in triplicate (one set for each culture) and 100 µl LB in triplicate

C. Measure fluorescence

1. Measure absorbance at 600 nm for all wells
2. Perform a single excitation at 480 nm and measure emission at 510 nm
3. Perform an emission scan: hit at 480 nm and measure from 500-580 nm
4. Perform an excitation scan: excite from 400-489 nm and measure at 510 nm

D. Analyze data

• Normalize for cell density
  • Did you observe a signal above background?
• Look at the emission and excitation scans for the cells: do they look like purified GFP?
• Plot excitation and emission scans on same graph
• Calculate GFP spectrum for cells + I13522 (subtract spectrum for cells + R0040)

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 21 November 2007
Updated 13 November 2013