Day 0

Streak out frozen stocks of two strains, which consist of a biobrick (K592009), vector (K608002), and type of E. coli (TOP10 and MDS42), on separate plates.

1. Find two LB/CAM plates from the cold room (4°C) and a bench with a Bunsen burner
2. Get ice bucket and fill with ice
3. Collect previously made frozen glycerol stocks (TOP10 and MDS42) from the -80°C freezer and place them on ice
4. Quickly return to your bench and demonstrate sterile technique (sterilize bench top with 70% EtOH and then light your bunsen burner)
5. Sterilize an inoculation loop (immerse in 70% EtOH and flame until the loop is visibly red hot)
6. Use the inoculation loop to streak out your cells onto each plate (refer to Lab 2 reading streaking procedure). Flame in between streakings.
7. Flip the plates upside down and place them in the 37°C incubator overnight

Day 1

Your plates will need to grow in the incubator for at least 20 hours.

Record/document observations of your plates. How many colonies? Colors? Are they isolated? Anything interesting? (take pictures)

Pick six independent colonies from each plate that appear to maintain the desired phenotype and start six 5 ml overnight cultures in LB/CAM media for each strain.

1. Sterilize your bench and turn on the Bunsen burner
2. Collect 12 culture tubes and label them (Day 1, your initials, date, strain, and colony #1-6)
3. Fill each culture tube with 5ml of LB/CAM culture using a disposable glass pipette
4. If your protein has a distinct color, choose colonies that visibly show that color. This is not guaranteed for all devices, since very unstable devices may already be broken. If there aren’t enough colonies with the desired phenotype, make a new plate.
5. Circle 6 chosen colonies with a sharpie on each plate
6. How to pick the colonies:

1. Using a sterile p1000 tip, gently pierce through the entire colony and agar
2. Place the entire tip into the culture tube. Slowly and gently pipette up and down to dislodge the agar and colony
3. Then, gently eject the tip so that it remains in the culture tube
4. Do this for all 6 chosen colonies

7. Repeat this for both strains
8. Place all culture tubes into the shaker incubator (37°C and at 200-225 RPM) overnight
9. Place the plates in the cold room (4°C) on the shelf labeled for your group

Day 2

Your cultures will need to grow in the incubator for at least 20 hours.

Record/document observations of your cultures. Color? Cloudy? Translucent? How many generations have passed? (take pictures)

Perform a 1:1000 dilution using LB/CAM media on each of your overnight cultures so that the growth starts once more from the first generation.

1. Turn on the Bunsen burner and use sterile technique
2. Find 12 microcentrifuge tubes and label them (1 for each O/N culture-strain and colony #)
3. Make sure the cultures are fully resuspended. If they were sitting in the cold room or a bench for more than 15 minutes, carefully vortex the culture until a homogenous mixture is created
4. Remove 250 µl from each overnight culture and place into its respective microcentrifuge tube
5. Start a fresh 5 ml culture for each O/N culture

1. Locate 12 new culture tubes and label them (Day 2, your initials, date, strain, and colony #1-6)
2. Pipette 5 ml of LB/CAM media into each culture tube using the glass pipette
3. You will be performing a 1:1000 dilution of the “Day 1” culture into the new media
4. Add 5 µl of each “Day 1” culture from its microcentrifuge tube into its respective new culture tube
5. Place these “Day 2” cultures back into the shaker incubator (37°C and at 200-225 RPM) overnight

6. Measure the chromoprotein absorbance for your cultures (intended color)

1. From the remaining culture in the microcentrifuge tube, add 200 µl to a well in a 96-well plate. There will be a single plate being used each day (ask Dr. Mishler)
2. Record what wells you will be pipetting into, and carefully add the 200 µl to just those wells. Avoid cross contamination.
3. Return the plate to a mentor/Dr. Mishler, who will store it in the cold room
4. The plate will be read by Dr. Mishler or a mentor later, once all of the samples for the day have been added

7. Clean up your bench space and store your overnight cultures from the previous day in the cold room on your group’s shelf

Day 3 → until completion

Repeat the procedure for Day 2, preparing new culture tubes with labeled with the respective days.

Record the absorbance readings from the previous day into your notebook, if they are available. This should be accessible online in our google drive folder.

Record how many generations passed between each day.

After 7-10+ days, or after a time when the color of the cultures visibly diminishes, you will be asked to stop carrying forward the overnight cultures. You will want to check this with Dr. Mishler or a mentor. In general, we will want to see that the color has declined and stayed declined for at least 2 consecutive days.

Day “completion” = Day X

In consultation with Dr. Mishler, stop growing cultures once the color has significantly diminished.

1. Plate a dilution of each O/N culture to generate single colonies

1. First, find 12 LB/CAM agar plates from the cold room. If there are none, then make some for your group.
2. Turn on the Bunsen burner and use sterile technique
3. Get 12 empty culture tubes (1 for each O/N culture) and label them according to their respective strain and colony #
4. Fill each of them with 5 ml of fresh LB/CAM broth using the glass pipette
5. Add 5 µl of each O/N culture to its respective new culture tube. This is a 1:1000 dilution.
6. Then, briefly vortex the cultures and let them sit for 1 minute.
7. Get 12 more empty culture tubes (1 for each culture) and label them once more, according to their respective strain and colony #
8. Fill each of them with 5 ml of fresh LB/CAM broth using the glass pipette
9. Add 5 µl of the 1:1000 dilution culture to its respective new culture tube. This is a 1:10^6 or a “1 to a million” dilution
10. Again, briefly vortex the cultures and let them sit for 1 minute
11. Label each LB/CAM plate with your initials, strain name, colony number, the date, and the day # of your culture tube
12. Plate 50 µl of the 1:10^6 dilutions onto an LB/CAM agar plate using a sterilized spreader. Do this for all 12 dilutions.
13. Place the plates into the 37°C incubator overnight

2. Prepare a glycerol stock for each final culture

1. Find 12 “cryo vials”
2. Label the lid of the vial with your initials, “BiB”, and the strain number (not name)
3. Label the side of the vial (white portion) with your initials, the strain name and colony #, the date, and the day # of the culture
4. Using sterile conditions, make glycerol stocks for each culture, the procedure for which can be found in the procedure manual.
5. Mix the glycerol stocks via vortexing/vigorous inverting
6. Place these stocks in the -80°C freezer, in a designated box

3. Final absorbance readings

1. Using your saturated O/N cultures, prepare a final set of of absorbance readings. Add your cultures to the 96-well plate as you previously have done

4. Take a picture of your cultures from Day 1→ today

1. Find your cultures in the cold room
2. Arrange these cultures in day order and a take a picture for all 6 sets for each strain

Day X+1

1. Remove your plates from the incubator and take a picture of them side by side with your original day 0 plate (find in cold room)
2. Record your observations of the plates in your lab notebook
3. Pick 1 colony from each of your 12 plates

1. This colony should not be strongly colored
2. This colony should be representative of the majority of colonies on the plate
3. Pick a random colony, and mark each chosen colony with a sharpie on the back of the plate

4. Turn on a Bunsen burner and use sterile technique
5. Locate 12 culture tubes label them for each strain and colony #
6. Fill them each with 5 ml of LB/CAM broth using a glass pipette
7. Gently touch each chosen colony with a red pipette tip and drop the tip into its respective culture tube
8. Place these tubes in the shaker incubator (37°C and at 200-225 RPM) overnight

Day X+2

1. Prepare a glycerol stock for each colony culture

1. Find 12 “cryo vials”
2. Label the lid of the vial with your initials, BiB, strain number followed by “C” (the “C” stands for colony)
3. Label the side of the vial (white portion) with your initials, the strain name, colony number, the date, the day # of the culture, and “colony”
4. Make the glycerol stock using the previously described procedure
5. Mix via vortexing/vigorous inverting
6. Store in the -80°C freezer, in a designated box

2. Miniprep each O/N culture and send them out for sequencing

1. Miniprep the six O/N cultures using the procedure given in the miniprep kit
2. Before you begin the manual procedure, you will need to vortex the culture
3. Upon completing the miniprep process, you will elute into a fresh microcentrifuge tube. Label each:

1. On lid- “Mini”, your initials, BiB, and strain number followed by “C”
2. On side- your initials, the date, culture #, and day #

4. Nanodrop these samples and record the concentrations on the side of the tube and in your notebook.
5. Prepare the samples for sequencing according to their concentrations. Follow the procedure given in the Lab Procedures/Protocols manual

-- Main.SeanLeonard - 25 Aug 2016