IGEM:MIT/2006/Notebook/2007-8-23

=J45200/J45250 3-Hour Pulse experiment=

Protocol

Protocol for 3-hour pulse:

1. Add the following in a 2-L flask:


 * 275 mL LB AMP/TET


 * 27.5 uL grown J45120 culture

2. Add the following in a 2-L baffled flask:


 * 275 mL LB AMP/TET


 * 27.5 uL grown J45181 culture

3. Grow both cultures at 220 RPM at 37C, starting J45200 20 minutes before J45250. Once the culture is put at 110 RPM and 37C, it is Time Zero

4. After x hours (for x=4 to x=12), extract 25 mL culture from master culture, pour into 125-mL flask, and add 13.6 uL to it for both J45200 and J45250 (grow up at 110 RPM)

5. At hour x+3 for hour x, GC extract culture

=Method 11/30=

Started Indole-Knockout and Top10 25-mL cultures (in 250-mL baffled flasks at 37C at 220 RPM) to be GC extracted tomorrow- 5:30 PM

IK- 20 hr growth, OD600=3.96

Top10- 20 hr growth, OD600= 3.34

=Additional Extractions- 1/13=

With old cultures, GC extracted culture spiked with A) .457 uL IAC B) .571 uL IAC C) 1.168 uL IAC--- will see extraction efficiency- extracted 200 uL, 200 uL heptane also added

For GC vials, 150 uL of each sample added with 250 uL heptane.

Also, standards were made.

.467 uL 1:10 octyl acetate added to 400 uL samples.

NOTE- 200640 has ten times as much octyl acetate as desired.

=Repeat 10/14=

Different timing:

Time Zero for J45200- 11:30

Time Zero for J45250- 12:30

Pulse for J45200 at 3:10, 3:30, 3:50, 6:10, 6:30, 6:50

Pulse for J45250 at 4:10, 4:30, 4:50, 7:10, 7:30, 7:50

GC extracts for J45200 at 6:10, 6:30, 6:50, 9:10, 9:30, 9:50

GC extracts for J45250 at 7:10, 7:30, 7:50, 10:10, 10:30, 10:50

=GC vial protocol=

1. Add 250 uL heptane to GC vial

2. Add 150 uL sample to GC vial

3. Add 2.335 1:50 octyl acetate for 400 uL samples

4. For 500, 200, 100, 25, and 5 PPM samples, add 3.504 uL, 2.92, 4.38, 4.672, and 5.84 uL octyl acetate

=Repeat 1/17=

Time Zero for J45200- 8:05

Time Zero for J45250- 9:25

Pulse for J45200- 1:05, 1:25, 1:45, 2:05

Pulse for J45250- 2:25, 2:45, 3:05, 3:25

GC extracts for J45200- 4:05, 4:25, 4:45, 5:05

GC extracts for J45250- 5:25, 5:45, 6:05, 6:25

OD600s

Before IA Addition

J45200

5:00- 0.20

5:20- 0.30

5:40- 0.38

6:00- 0.54

J45250

5:00- 0.34

5:20- 0.48

5:40- 0.68

6:00- 0.80

After IA Addition

J45200

8:00- 0.84

8:20- 1.18

8:40- 1.24

9:00- 1.30

J45250

8:00- 1.30

8:20- 1.34

8:40- 1.46

9:00- 1.46

=Repeat 1/16=

Time Zero for J45200- 8:45

Time Zero for J45250- 10:05

Pulse for J45200- 12:25, 12:45, 1:05, 1:25

Pulse for J45250- 1:45, 2:05, 2:25, 2:45

GC extracts for J45200- 3:25, 3:45, 4:05, 4:25

GC extracts for J45250- 4:45, 5:05, 5:25, 5:45

OD600s

Before IA Addition

J45200

3:40- 0.04

4:00- 0.04

4:20- 0.08

4:40- 0.14

J45250

3:40- 0.04

4:00- 0.08

4:20- 0.20

4:40- 0.28

After IA Addition

J45200

6:40- 0.16

7:00- 0.20

7:20- 0.38

7:40- 0.56

J45250

6:40- 0.26

7:00- 0.44

7:20- 0.66

7:40- 1.12

=Repeat 1/11=

Time Zero for J45200- 6:00

Time Zero for J45250- 8:00

Pulse for J45200- 10:20, 10:40, 11:00, 11:20, 11:40

Pulse for J45250- 12:20, 12:40, 1:00, 1:20, 1:40

GC extracts for J45200- 1:20, 1:40, 2:00, 2:20, 2:40

GC extracts for J45250- 3:20, 3:40, 4:00, 4:20, 4:40

OD600s

Before IA Addition

J45200

4:20- 0.08--- MISLABELED

4:40- 0.12

5:00- 0.20

5:20- 0.26

5:40- 0.38

J45250

4:20- 0.18--- MISLABELED

4:40- 0.28

5:00- 0.38

5:20- 0.54

5:40- 0.72

J45200

7:20- 0.44--- MISLABELED- DO NOT USE

7:40- 0.48

8:00- 0.66

8:20- 0.90

8:40- 1.20

J45250

7:20- 0.40--- MISLABELED- DO NOT USE

7:40- 1.14

8:00- 1.26

8:20- 1.38

8:40- 1.42

=Repeat 1/9=

Time Zero for J45200- 5:15

Time Zero for J45250- 6:15

Pulse for J45200- 8:55, 9:15, 9:35, 11:55, 12:15, 12:35

Pulse for J45250- 9:55, 10:15, 10:35, 12:55, 1:15, 1:35

GC extracts for J45200- 11:55, 12:15, 12:35, 2:55, 3:15, 3:35

GC extracts for J45250- 12:55, 1:15, 1:35, 3:55, 4:15, 4:35

OD600s for 1/9

Before IA Addition

J45200

3:40- 0.04

4:00- 0.04

4:20- 0.08

6:40- 0.98

7:00- 1.10

7:20- 1.26

J45250

3:40- 0.04

4:00- 0.08

4:20- 0.14

6:40- 1.28

7:00- 1.46

7:20- 1.54

After IA Addition

J45200

6:40- 0.16

7:00- 0.18

7:20- 0.40

9:40- 1.48

10:00- 1.50

10:20- 1.56

J45250

6:40- 0.24

7:00- 0.38

7:20- 0.64

9:40- 1.70

10:00- 1.72

10:20- 1.74

OD600s

Before Isoamyl Alcohol Addition

J45200

3:40- 0.06

4:00- 0.06

4:20- 0.08

6:40- 0.80

7:00- 0.98

7:20- 1.18

J45250

3:40- 0.04

4:00- 0.06

4:20- 0.10

6:40- 1.04

7:00- 1.24

7:20- 1.46

After Isoamyl Alcohol Addition

J45200

6:40- 0.16

7:00- 0.26

7:20- 0.42

9:40- 1.64

10:00- 1.38

10:20- 1.46

J45250

6:40- 0.18

7:00- 0.24

7:20- 0.48

9:40- 1.62

10:00- 1.86

10:20- 1.88

=Side experiment=

Picked colonies from fresh plates of 600 (OD600=1.82, Time=21 hrs.) and 900 (LOST SAMPLE), will grow up 1 L culture (in 2500 mL baffled flasks at 220 RPM) and GC extract using 1-L separatory funnel GC extract: 2 mL per 1 L media (15 mins separation time)

Redid 900 (OD600=2.16, Time=50 hrs.)

Redo of 900 (OD600=2.72, Time=21 hrs.) and 200

Redo of 900 (OD600=2.22, Time = 19 hrs.) and 200 (OD600=2.48, Time = 19 hrs.) and 900 (OD600=1.94, Time = 19 hrs.)

=Other side experiment=

Two GC extract efficiency GC extractions

- 200 ppm (4.56 uL) IAc in J45200 culture (OD600= 2.16)

- 100 ppm (2.28 uL) IAc in J45250 culture (OD600= 1.82)

=OD600s (Repeat 10/13)=

-Start at 12:07 for 200 and 12:27 for 250

Before Addition of Isoamyl Alcohol

J45200

2- 0.00

3- 0.00

4- 0.04

5- 0.16

6- 0.42

7- 0.98

J45250

2- 0.00

3- 0.00

4- 0.06

5- 0.18

6- 0.52

7- 1.10

After Addition of Isoamyl Alcohol

J45200

5- 0.00

6- 0.04

7- 0.16

8- 0.60

9- 1.28

10- 1.42

J45250

5- 0.02

6- 0.04

7- 0.18

8- 1.02

9- 1.48

10- 1.80

=Side experiment (10/13)=

-Make 50 mL, 250 uL culture of 181 AT3, 250 AC3 and grow at 220 RPM for 20 mins (12:07)

-Add isoamyl alcohol and salicylic acid (standard amounts) and split cultures

-GC extract one in exponential (OD600 = 0.32, WORRIED) and one in stationary phase (OD600 = 2.24)

=Other Side experiment (10/13)=

-Took J45200 after 7 hrs and added 2.28 uL isoamyl acetate to 20 mL culture (100 ppm IAc) to get a data pt for extraction efficiency

=OD600s (Repeat 9/29)=

Before Isoamyl Alcohol Addition

J45200

4 hours- 0.12

5 hours- 0.34

6 hours- 0.76

7 hours- 1.30

8 hours- 1.92

9 hours- 2.14

10 hours- 2.44

11 hours- 2.52

J45250

4 hours- 0.18

5 hours- 0.50

6 hours- 1.14

7 hours- 1.58

8 hours- 1.92

9 hours- 2.06

10 hours- 2.24

11 hours- 2.34

After Isoamyl Alcohol Addition

J45200

7 hours- 0.46

8 hours- 1.28

9 hours- 1.48

10 hours- 1.68

11 hours- 1.92

12 hours- 2.12

13 hours- 2.40

14 hours- 2.68

J45250

7 hours- 1.00

8 hours- 1.26

9 hours- 1.74

10 hours- 1.92

11 hours- 1.98

12 hours- 2.10

13 hours- 2.26

14 hours- 2.44

=OD600s=

Before Isoamyl Alcohol Addition

J45200

4 hours- 0.06

5 hours- 0.18

6 hours- 0.54

7 hours- 1.02

8 hours- 1.60

9 hours- 2.04

10 hours- 2.32

11 hours- 2.54

12 hours- 2.76

J45250

4 hours- 0.08

5 hours- 0.26

6 hours- 0.66

7 hours- 1.44

8 hours- 1.70

9 hours- 1.84

10 hours- 2.08

11 hours- 2.16

12 hours- 2.30

After Isoamyl Alcohol Addition

J45200

7 hours- 0.34

8 hours- 1.08

9 hours- 1.34

10 hours- 1.58

11 hours- 1.76

12 hours- 2.06

13 hours- 2.40

14 hours- 2.64

15 hours- 2.88

J45250

7 hours- 0.46

8 hours- 1.34

9 hours- 1.52

10 hours- 1.82

11 hours- 1.88

12 hours- 2.00

13 hours- 2.16

14 hours- 2.24

15 hours- 2.42

=Other Things to Do=

1. Swing by Stephanopoulos lab to see status of the GC- DONE (bad news: delayed, needs until Saturday to set it up; good news: nobody is set up to use it, so I should be able to use it all of next week)

2. At 4:40, dilute the J45995, J45996, I7100, and B0015 cultures 1:250- SCRAP THIS (NOT ENOUGH FLASKS)

3. Ask grad students about next step in wintergreen curve production- DONE (go with gut instinct)