Protocol for Lube Control Experiment

1. Blank spectrophotometer (using LB).
2. Measure pre-inoculation OD (see table below)
3. Get 12 x 50ml Falcon tubes
4. Label Tubes (1-6) as follows: %Lube, Control E.Coli W3110, iGEM, MA, Date
5. Label Tubes (7-12) as follows: %Lube, Media Only, iGEM, MA, Date
6. Get lube and LB
7. Arrange solution in each tube (1-6) as following:

Tube	% Lube	ml Lube	ml LB
1	0	0	30
2	20	6	24
3	40	12	18
4	60	18	12
5	80	24	6
6	100	30	0

8. Mix solution by inverting and ensure that everything is mixed
9. Remove 20ml of each of the solutions in tubes 1-6 and add each to its corresponding Lube% tube (tubes 7-12).
10. Put tubes 7-12 to the side as they will be used later.
11. To each of the tubes 1-6 add 200ul of the prepared glycerol stock (prepared as described in handbook).
12. Prepare 12 cuvettes for spectrophotometer.
13. To each cuvette add 750ul of media from tubes 7-12 (ie. cuvette 1 gets 0% lube media, cuvette 2 gets 20% lube media, etc.) (As we are doing replicates you will have duplicates of all cuvettes. Do not mix up)
14. Add 250ul of broth to its corresponding cuvette (according to lube concentration).
15. Add tubes 1-6 to 37°C shaker.
16. Start timer for 30 minutes.
17. Now check OD of all of the cuvettes. Record data in table given below.
18. Repeat all of these steps until the table below is full (or ideally from 9.00- 17.00)

Raw Data Processing: As the cuvette samples are diluted we need to adjust that before processing the data. This can be done with this formula.

MeasuredOD × 4=Actual OD

Protocol for NarK-Lycopene induction Experiment

Previously nitrogen gas was used to induce the anaerobic conditions needed for inducing expression of NarK.

We tried three different inducers for NarK (the inducible promoter for lycopene growth:

1. Sodium nitrite

2. Sodium nitrate

3. Sodium nitroprusside (SNP), which is a nitric oxide donor.

These were made up to 1M stock solutions.

Our Protocol:

1. Pick 3 colonies from a plate and grow them up in 5ml of LB and antibiotic overnight in a shaker at 37C to give you culture 1, 2 and 3.

2. The next morning, prepare 7 tubes with 5mls of fresh LB and antibiotic for each culture, 1,2 and 3.

This will give you enough for 3 replicates for each concentration plus an uninduced negative control.

3. Inoculate the 7 fresh tubes with 5ul from the respective overnight culture and grow for 3 hours in a shaker at 37C.

4. Take the cultures out and add the appropriate amount of 1M stock inducer to give the concentrations below for each reagent, nitrite, nitrate and SNP.

0uM

1uM

10uM

100uM

1mM

10mM

100mM

5. Grow overnight at 37C in a shaker with loose caps and measure induction in the morning (so roughly 16 hours).

6. Follow the protocol below for how we assayed lycopene production.

7. Repeat with smaller increments of concentration around the concentration of which lycopene is induced.

DAY 1
1) 5pm use 100uL of ROG1 glycerol stock to inoculate 8mL of LB media containing 100 μg/L Chloramphenicol in a 15mL Falcon tube.


2) Split 4mL inoculum equally into four 15mL Falcon tubes, labeled


C1, C2, H1, H2


Loosen screw top lids and place in incubator shaker at 37C.


DAY 2


3) 9am tighten the lids of tubes H1 and H2 and seal around lids with parafilm. Return to incubator shaker at 37C.


4) 3pm centrifuge 15mL Falcon tubes at 4000 rpm for 10 minutes.


5) Re-suspend cells in 300uL acetone, transfer to pre-labelled 1.5mL Eppendorf tubes and vortex for 5 minutes.


6) Centrifuge sample at 14,000 rpm for 1 minute. Collect supernatant. Store in 4C fridge and proceed to step 7 when convenient.


7) Measure absorption at 450 nm using Microplate Reader.