Team:Acton-BoxboroughRHS/Labs

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Project Lab
empty 3A Assembly Kit (igem)

The 3A Assembly Kit and following protocols will take you through the process of 3A Assembly, and by the end you will have assembled your own composite part in the lab. The kit includes two parts: Part A (BBa_J04500) and Part B (BBa_J04650), which when assembled together will form a RFP (red fluorescent protein) generator. Your cells will turn red!

Step 1: Growing the E. coli

Streaking the Agar Stabs

(About 15 active minutes and 16-24 hours of incubation are needed.)

• 70% ethanol
• Paper towels
• Lab marker/Sharpie
• Agar Stab: Part A - BBa_J04500 (kit)
• Agar Stab: Part B - BBa_J04650 (kit)
• Inoculating loops (kit)
• LB agar plates - Amp/Kan (kit)

1. Clean the lab bench by wiping down with 70% ethanol and paper towels.
2. Part A (BBa_J04500) and Part B (BBa_J04650) are both maintained on pSB1AK3 plasmid backbones, which means they are ampicillin- and kanamycin-resistant. Label the agar plates with the names of Part A and Part B.
3. Notice how each zig-zag overlaps with the previous one just a little, and only at the end.
4. Use an inoculating loop to transfer some cells from the Part A agar stab to the appropriately labeled Amp/Kan agar plate. There is a hole in each agar stab from where it was inoculated. Dip an inoculating loop into the stab at the same location, and streak the bacteria onto the agar plate in a zig-zag pattern. Using a fresh inoculating loop, streak onto the agar plate again creating a new zig-zag pattern that overlaps the first. This will help ensure that you will have single colonies to pick from. Streak gently, and try not to puncture the agar.
5. Repeat step 4 for Part B..
6. This prevents other bacteria from settling, and growing, on your agar plate.
7. Place the agar plates into the incubator with the agar side facing up, lid facing down (see insert). Incubate the agar plates at 37° C for 14-16 hours. Alternately, incubate at room temperature for 24-30 hours.
8. Once your agar plates have grown up you can store them in your fridge (4° C) until you're ready to grow up your cell culture.
9. Plates can be stored at 4° C for up to 3 weeks.

Growing up Cell Cultures

(About 30 active minutes and 16 hours of incubation are needed.)

• 70% ethanol
• paper towels
• Lab marker/Sharpie
• 14ml culture tubes (kit)
• 10ml of LB broth - Amp/Kan (kit)
• Inoculating loops (kit)
• Agar plate: Part A - BBa_J04500 (see previous step)
• Agar plate: Part B - BBa_J04650 (see previous step)
• Rotator/Shaker

1. Clean the lab bench by wiping down with 70% ethanol and paper towels.
2. Remove the agar plates for Part A and Part B from the incubator or 4° C fridge.
3. Label one 14ml culture tube for each Part. Add 5ml of LB broth (with ampicillin and kanamycin) to each culture tube.
4. Use an inoculating loop to pick a single colony from each agar plate and inoculate the LB broth, in the appropriately labeled culture tube. Do not use the same inoculating loop more than once! Press lightly on the snap caps of the 14ml tubes, the caps should be a bit loose to allow for air flow.
5. Incubate for 16 hours at 37° C, in a rotator or shaker. Rotation helps the cells grow faster, and prevents them from settling at the bottom.
6. After incubation, the cell culture should be cloudy. You can now firmly press down on the snap caps to seal the tubes and store the cell culture at 4° C until you're ready to move onto the next step.

Step 2: Miniature Preparations

(About 1 active hour is needed.)

• 70% ethanol
• Paper towels
• Marker/Sharpie
• Centrifuge/microcentrifuge
• 2 Beakers/waste collection containers
• Bleach
• Buffers: P1, RNAse A, P2, N3, PB, PE (kit)
• 1.7ml Microcentrifuge tubes (kit)
• Qiagen spin columns (kit)
• Distilled water (kit)
• Cell culture: Part A - BBa_J04500 (see previous step)
• Cell culture: Part B - BBa_J04650 (see previous step)
• Nanodrop machine (optional)

1. Note: If you haven't already, add the RNAse A to both Buffer P1 aliquots. Once you add RNAse A, you must store Buffer P1 at 4° C until use.
2. Clean the lab bench by wiping down with 70% ethanol and paper towels.
3. Make sure the cap of the culture tubes creates a firm seal. Spin down the two culture tubes in a centrifuge. Spin for 3 minutes at 8000 rpm.
4. You should see a pellet of collected cells at the bottom of the tube. Slowly pour the supernatant (media at the top of the tube) into the beaker. Be sure not to do it too quickly or the cell pellet might dislodge. Pour some bleach into the beaker to sterilize the solution.
5. Pipet 250ul of Buffer P1 to each 14ml culture tube. Pipet up and down gently to re-suspend the cell pellets.
6. Label one 1.7ml microcentrifuge tube each with the part name. Transfer the resuspended cells to the appropriate microcentrifuge tube.
7. Pipet 250ul of Buffer P2 to each microcentrifuge tube. Close the tubes, and flip them upside down gently 5x.
8. Carefully open the tubes, and pipet 350ul of Buffer N3 into each one. Close the tubes, and flip them upside down gently 5x to mix. The solution will turn clear and slightly “chunky”.
9. Spin down the samples in a microcentrifuge for 10 minutes at 13,000 rpm. This spin will separate cellular debris onto the side of the tubes.
10. Label one Qiagen spin column for each part. Carefully pipet the supernatant (the clear liquid) to the appropriate spin column. Try not to transfer the white debris pelleted onto the side of the tubes.
11. Spin down the spin column tubes. In a microcentrifuge, spin for 1 minute at 13,000 rpm.
12. Remove the filter tube (top) from the collection tube (bottom). Be sure not to confuse the two samples! Pour the flow-through in the collection tubes into the other beaker. Place the filter tube back into its original collection tube.
13. Pipet 500ul Buffer PB to each spin column. Spin down the samples. In a microcentrifuge, spin for 1 minute at 13,000 rpm.
14. Remove the filter tube from the collection tube. Be sure not to confuse the two samples! Pour the flow-through into the other beaker. Place the filter tube back into its original collection tube.
15. Pipet 750ul Buffer PE to each spin column. Spin down the samples. In a microcentrifuge, spin for 1 minute at 13,000 rpm.
16. Remove the filter tube from the collection tube. Be sure not to confuse the two samples! Pour the flow-through into the other beaker. Place the filter tube back into its original collection tube.
17. The spin columns should appear empty, but spin down the samples for 1 minute at 13,000 rpm again. This will remove any remaining buffer from the filter tubes.
18. Label one clean 1.7ml microcentrifuge tube each with a part name. Transfer the appropriate filter tube to the clean 1.7ml tube.
19. Pipet 50ul of distilled water to the center of each filter tube. Let the samples sit for 1 minute, then spin down the samples. In a microcentrifuge, spin for 1 minute at 13,000 rpm.
20. If you can, calculate the concentration of the DNA, and mark this on the microcentrifuge tubes. They can be stored at -20° C for long-term storage, or at 4° C for short-term storage.
1. To determine the concentration of DNA using a Nanodrop, open the Nanodrop program on the attached computer.
2. Select the "Nucleic Acid" option. Pipet 2ul of distilled water onto the bottom sensor. Gently close the top arm, and click "OK" to activate the machine. After the machine has finished activating, click on "Blank".
3. Lift the arm. Wet a Kimwipe with distilled water, and use this to gently wipe the top and bottom sensor clean.
4. Pipet 2ul of your miniprepped Part A DNA sample onto the bottom sensor, then click on "Measure". Write down the concentration on the side of the tube.
5. Repeat steps 3-4 for your miniprepped Part B DNA sample.
6. Lift the arm. Wipe the top and bottom sensor with the damp Kimwipe.

Step 3: Restriction Digest

(About 30 active minutes and 50 minutes of incubation are needed.)

• 70% ethanol
• Paper towels
• Ice
• Container for ice
• Lab marker/Sharpie
• pSB1C3 linearized plasmid backbone (25ng/ul) (kit)
• Part A (25ng/ul) (kit)
• Part B (25ng/ul) (kit)
• RFP Control (20ng/ul) (kit)
• 0.6ml tubes (kit)
• NEB buffer 2
• BSA
• NEB enzymes: EcoRI, SpeI, XbaI, PstI
• Thermocycler, or waterbath and thermometer

1. Clean the lab bench by wiping down with 70% ethanol and paper towels.
2. Keep all enzymes and buffers used in this section on ice.
3. Thaw NEB Buffer 2 and BSA in room temperature water. Re-homogenize both by inverting the tubes, and flick/spin them to collect the liquid at the bottom of the tube.
4. Label four 0.6 tubes: Part A, Part B, pSB1C3 (linearized plasmid backbone), and RFP Control
5. Add 500ng of DNA to the appropriate tube. Add distilled water to the tubes for a total volume of 42.5ul in each tube.
6. Pipet 5ul of Buffer 2 to each tube.
7. Pipet 0.5ul of BSA to each tube.
8. In the Part A tube: Add 1ul of EcoRI enzyme, and 1ul of SpeI enzyme.
9. In the Part B tube: Add 1ul of XbaI enzyme, and 1ul of PstI enzyme.
10. In the pSB1C3 tube: Add 1ul of EcoRI enzyme, and 1ul of PstI enzyme.
11. In the RFP Control tube: Add 1ul of EcoRI enzyme, and 1ul of PstI enzyme.
12. The total volume in each tube should be approximately 50ul. Mix well by pipetting slowly up and down 5x. Be gentle, and do not vortex. Spin the samples for 5 seconds in a microcentrifuge, or flick them to collect all of the mixture to the bottom of the tube.
13. Incubate the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes. We use a thermocycler, but a waterbath and an accurate thermometer works well also!
14. The digested DNA can be stored at 4°C for a few days. For longer storage, keep at -20°C.

Step 4: Ligation

(About 15 active minutes and 50 minutes of incubation are needed.)

• 70% ethanol
• Paper towels
• Lab marker/Sharpie
• 0.6 tubes (kit)
• Distilled water (kit)
• Ice
• Container for ice
• T4 DNA Ligase Reaction Buffer
• T4 DNA Ligase
• Microcentrifuge
• Thermocycler, or waterbath and thermometer
• Restriction Digest: pSB1C3 linearized plasmid backbone (see previous step)
• Restriction Digest: Part A (see previous step)
• Restriction Digest: Part B (see previous step)
• Restriction Digest: RFP Control (see previous step)

1. Clean the lab bench by wiping down with 70% ethanol and paper towels.
2. Thaw T4 DNA Ligase Reaction Buffer at room temperature. Keep the T4 DNA Ligase in the freezer until you're ready to use it.
3. Label one 0.6ml tube as New Part.
4. Add 2ul from the pSB1C3 linearized plasmid backbone digest.
5. Add 3.3ul from the Part A digest.
6. Add 3.9ul from the Part B digest.
7. Add 1ul of T4 DNA Ligase Reaction Buffer.
8. Add 0.5ul of T4 DNA Ligase (keep this at -20°C until use!).
9. Mix by gently pipetting up and down 3x. Do not vortex; this inactivates the enzymes. Place tube in microcentrifuge for a quick 5 second spin or flick the tube to collect the mixture at the bottom.
10. Label one 0.6 tube as Ligation Control.
11. Add 2ul from the RFP Control digest.
12. Add 6.5ul of distilled water.
13. Add 1ul of T4 DNA Ligase Reaction Buffer.
14. Add 0.5ul of T4 DNA Ligase.
15. Mix by gently pipetting up and down 3x. Do not vortex; this inactivates the enzymes. Place tube in microcentrifuge for a quick 5 second spin or flick the tube to collect the mixture at the bottom.
16. Incubate at 16°C for 30 minutes, then at 80°C for 20 minutes. We use a thermocycler, but a waterbath and thermometer combination works great too! The ligated products can be stored at -20°C.

Step 5: Transformation

(About 1 active hour and 12-24 hours of incubation are needed.)

• 70% ethanol
• Paper towels
• Lab marker/Sharpie
• Ice
• Container for ice
• Timer
• NEB10 cells (see Growing step for preparation instructions)
• 2.0ml microcentrifuge tubes (kit)
• Inoculating loops (kit)
• Ligation: New Part (see previous step)
• Ligation: Control (see previous step)
• DNA: RFP Control (20ng/ul) (kit)
• SOC media (kit)
• LB agar plates - Chloramphenicol (kit)
• Sterile glass beads (kit)
• Waterbath and thermometer

1. Note: SOC media gets contaminated easily, so be careful when handling. if possible, wear gloves, and only open the container when you need to.
2. Clean the lab bench by wiping down with 70% ethanol and paper towels.
3. Keep all materials on ice unless otherwise specified! This will help make the cells more competent and easier to transform.
4. Label a 2.0ml microcentrifuge tube as Transformation Control, another as Ligation: New Part, and one more as Ligation Control.
5. Add 5ul of RFP Control DNA (20ng/ul) into the Transformation Control tube.
6. Add 2ul of the New Part ligation product into the Ligation: New Part tube.
7. Add 2ul of the RFP Control ligation product into the Ligation Control tube.
8. Place the tubes on ice to pre-chill them.
9. Thaw one competent cell aliquot tube on ice (this takes about 5-8 minutes).
10. Gently flick the tube of competent cells, then pipet 50ul of competent cells into each 2.0ml microcentrifuge tube.
11. Try to keep the cells as cold as possible by holding just the top of the tube, not the bottom where the cells are.
12. Incubate the DNA and cell mixtures on ice for 30 minutes. During this incubation, pre-heat the waterbath to 42°C.
13. Place the tubes into the waterbath for 60 seconds. Immediately place the tubes back on ice for 5 minutes.
14. Add 200ul of SOC media to each tube. Gently tap the tubes with your finger to mix.
15. Incubate the tubes at 37°C for 2 hours. During this time, prepare the agar plates by labeling them. Add 3 - 6 glass beads per plate.
16. Pipet 200ul of the Transformation Control onto the appropriate plate. Spread evenly over the surface of the agar by gently shaking the plate back and forth. The beads will do the work for you!
17. Repeat step 11 for the other two transformations.
18. Place the agar plates into the incubator with the agar side facing up, lid facing down. Incubate the agar plates at 37°C for 12 - 14 hours. Alternately, incubate at room temperature for 24 hours.
19. Check for red colonies the next day, and post your results online in the next section! Plates can be stored at 4° Celsius for up to two weeks.

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