iGEM 2018 — Team CCA-San_Diego

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# Team:CCA-San Diego/Notebook

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* Project

  [Description](https://2018.igem.org/Team%3ACCA-San_Diego/Description)
  [Design](https://2018.igem.org/Team%3ACCA-San_Diego/Design)
  [Parts](https://2018.igem.org/Team%3ACCA-San_Diego/Parts)
  [Results](https://2018.igem.org/Team%3ACCA-San_Diego/Results)
  [Applied Design](https://2018.igem.org/Team%3ACCA-San_Diego/Applied_Design)
  [Entrepreneurship](https://2018.igem.org/Team%3ACCA-San_Diego/Entrepreneurship)
* Experiments

  [Experiments](https://2018.igem.org/Team%3ACCA-San_Diego/Experiments)
  [Measurement](https://2018.igem.org/Team%3ACCA-San_Diego/Measurement)
  [Model](https://2018.igem.org/Team%3ACCA-San_Diego/Model)
  [Demonstrate](https://2018.igem.org/Team%3ACCA-San_Diego/Demonstrate)
  [Notebook](https://2018.igem.org/Team%3ACCA-San_Diego/Notebook)
  [Interlab](https://2018.igem.org/Team%3ACCA-San_Diego/Interlab)
  [Safety](https://2018.igem.org/Team%3ACCA-San_Diego/Safety)
* Parts

  [Parts](https://2018.igem.org/Team%3ACCA-San_Diego/Parts)
  [Basic Part](https://2018.igem.org/Team%3ACCA-San_Diego/Basic_Part)
  [Composite Part](https://2018.igem.org/Team%3ACCA-San_Diego/Composite_Part)
  [Part Collection](https://2018.igem.org/Team%3ACCA-San_Diego/Part_Collection)
  [Improve](https://2018.igem.org/Team%3ACCA-San_Diego/Improve)
* Human Practices

  [Human Practices](https://2018.igem.org/Team%3ACCA-San_Diego/Human_Practices)
  [Engagement](https://2018.igem.org/Team%3ACCA-San_Diego/Engagement)
* Team

  [Team](https://2018.igem.org/Team%3ACCA-San_Diego/Team)
  [Attributions](https://2018.igem.org/Team%3ACCA-San_Diego/Attributions)
  [Collaborations](https://2018.igem.org/Team%3ACCA-San_Diego/Collaborations)


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# Team:CCA-San Diego/Experiments

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* Project

  [Description](https://2018.igem.org/Team%3ACCA-San_Diego/Description)
  [Design](https://2018.igem.org/Team%3ACCA-San_Diego/Design)
  [Parts](https://2018.igem.org/Team%3ACCA-San_Diego/Parts)
  [Results](https://2018.igem.org/Team%3ACCA-San_Diego/Results)
  [Applied Design](https://2018.igem.org/Team%3ACCA-San_Diego/Applied_Design)
  [Entrepreneurship](https://2018.igem.org/Team%3ACCA-San_Diego/Entrepreneurship)
* Experiments

  [Experiments](https://2018.igem.org/Team%3ACCA-San_Diego/Experiments)
  [Measurement](https://2018.igem.org/Team%3ACCA-San_Diego/Measurement)
  [Model](https://2018.igem.org/Team%3ACCA-San_Diego/Model)
  [Demonstrate](https://2018.igem.org/Team%3ACCA-San_Diego/Demonstrate)
  [Notebook](https://2018.igem.org/Team%3ACCA-San_Diego/Notebook)
  [Interlab](https://2018.igem.org/Team%3ACCA-San_Diego/Interlab)
  [Safety](https://2018.igem.org/Team%3ACCA-San_Diego/Safety)
* Parts

  [Parts](https://2018.igem.org/Team%3ACCA-San_Diego/Parts)
  [Basic Part](https://2018.igem.org/Team%3ACCA-San_Diego/Basic_Part)
  [Composite Part](https://2018.igem.org/Team%3ACCA-San_Diego/Composite_Part)
  [Part Collection](https://2018.igem.org/Team%3ACCA-San_Diego/Part_Collection)
  [Improve](https://2018.igem.org/Team%3ACCA-San_Diego/Improve)
* Human Practices

  [Human Practices](https://2018.igem.org/Team%3ACCA-San_Diego/Human_Practices)
  [Engagement](https://2018.igem.org/Team%3ACCA-San_Diego/Engagement)
* Team

  [Team](https://2018.igem.org/Team%3ACCA-San_Diego/Team)
  [Attributions](https://2018.igem.org/Team%3ACCA-San_Diego/Attributions)
  [Collaborations](https://2018.igem.org/Team%3ACCA-San_Diego/Collaborations)

# DNA resuspension

DNA Preparation

1. Centrifuge the IDT tubes for 3-5 seconds at a minimum of 300 x g (.3 rcf) to ensure the material is in the bottom of the tube. [RCF = 1.12 x Radius x (rpm/1000)2]
2. Add TE buffer to reach a final concentration of 10ng/uL. (Label from bottle).

1. Construct 1 (\_\_\_\_\_fmol for 1000ng): Add 100 uL of Pure Water
2. Construct 2 (\_\_\_\_\_fmol for 1000ng): Add 100 uL of Pure Water
3. Construct 3 (\_\_\_\_\_fmol for 1000ng): Add 100 uL of Pure Water

3. Vortex Briefly → Flick or shake instead?
4. Incubate at 50˚C for 20 minutes. (Use PCR Machine)
5. Briefly Vortex and Centrifuge.

# Golden Gate Assembly

1. Prepare a mastermix by adding together

1. BsaI Part Digestion/Ligation
2. Enzyme (0.5 µL), T4 DNA Ligase (1.5 µL), 10X BSA (2 microliters, all one solution in kit)
3. 10X T4 Ligase Buffer (1.5 microliters in kit)
4. Deionized water (Up to 15 microliters)

2. Pipette up and down to mix
3. Distribute mastermix into the labeled PCR tube
4. Add DNA components for each reaction

1. [2,500/\_\_\_\_\_(from resuspension)]=\_\_\_\_ µL of Construct 1
2. [2,500/\_\_\_\_\_(from resuspension)]=\_\_\_\_uL of Construct 2
3. [2,500/\_\_\_\_\_(from resuspension)]=\_\_\_\_uL of Construct 3

5. Flick to mix
6. Place the reaction tubes into the thermocycler and adjust the settings for a given enzyme

|  |  |  |  |
| --- | --- | --- | --- |
| Repetition | Step | Temperature | Time |
| None | Initial Digestion | 37˚C | 10 min |
| Repeat 25X | Digestion | 37˚C | 1.5 min |
| Annealing and Ligation | 16˚C | 3 min |
| None | Digestion and Ligase inactivation | 50˚C | 5 min |
| Inactivation | 80˚C | 10 min |
| Storage | 12˚C | Infinite |

# DNA Purification

PCR Purification

1. Combine all reactions for all Golden Gate Digestions into one sample.
2. Dilute new sample with DNA Cleanup Binding Buffer according to the table below. Mix well by pipetting up and down or flicking the tube. Do not vortex.

1. We recommend a sample volume of 20-100 µl. For smaller samples, adjust the volume with TE. For diluted samples larger than 800 µl, load a portion of the sample, proceed with step 2, and repeat as necessary.

|  |  |  |
| --- | --- | --- |
| Sample Type | Ratio of Binding Buffer: Sample | Example |
| dsDNA>2 kb | 2:1 | 525.42uL:262.71uL  For Naphthalene  435.14uL:218.07uL  For HSU-ETCDR |

3. Insert column into collection tube and load sample onto column. Spin for 1 minute, then discard flow-through.
4. Re-insert column into collection tube. Add 200 µl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
5. Repeat step 3.
6. Transfer column to a clean 1.5 mL microfuge tube.

1. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute

7. Add 6 µl (greater than or equal to, but we want as small as possible so add 6uL) of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

1. Typical elution volumes are 6-20 µl. Nuclease-free water (pH 7-8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated. For larger size DNA (>= 10 kb), heating the elution buffer to 50℃ prior to use can improve yield.

# Assembly Protocol (Version 1)

Biobrick Digestion

1. Remove the DNA and the destination plasmid along with NEBuffer 2 and BSA from the freezer to thaw. It is recommended to thaw the tubes by immersing them in room temperature water or remove them from the freezer and leave them in a cold box.
2. One PCR tube is for digest of the DNA part, and the last one is for the destination plasmid. Be sure to label each tube (DNA, P)
3. To each tube, add H2O and 180 ng of the DNA and 180 ng (From plasmid kit) plasmid to be digested. Adjust the amount of water you add so the total volume in the DNA tube is 7.65 µl.
4. Add 0.9 µl of NEBuffer 2 to both tubes.
5. Add 0.09 µl of BSA to both tubes.
6. Add 0.18 µl of the EcoRI to both tubes.

1. Only touch the very end of the pipette tip into the restriction enzyme!!!

7. Add 0.18 µl of the PstI to both tubes.
8. The total volume should be 9 µl for both. Ensure the digest is well-mixed by flicking the tube. You can spin the tube in a microcentrifuge for a few seconds to collect the liquid at the bottom of the tube again.
9. Return all reagents and purified DNA to the -20C freezer

Incubation:

10. Incubate the two restriction digests at 37C for 30 min. A water bath, an incubator, or a thermocycler are suitable for this incubation.
11. Incubate the two restriction digests at 80C for 20 min to deactivate the restriction enzymes. This step is most conveniently performed in a thermocycler so choose tubes for the restriction digest that fit in your thermocycler. As an easy way to confirm the digests worked, consider running 20 µl of each digest on a 1% agarose gel and look for bands of the expected length.
12. Store the two restriction digests at -20C or proceed immediately to the ligation step.

Biobrick Assembly

1. Remove the 10X T4 DNA Ligase Reaction Buffer from freezer to thaw. T4 DNA Ligase enzyme can either be placed in a cold box to keep it close to -20C or immerse buffer tube in room temperature water. Once thawed, agitate the 10X T4 DNA Ligase Reaction Buffer until all precipitate goes into solution
2. Add 2.75 µl of H2O to a 200 µl PCR tube.
3. Add 1 µl from the DNA digest and 0.5 µl from the plasmid digest to the tube.
4. Add 0.5 µl of 10X T4 DNA Ligase Reaction Buffer to the tube
5. Add 0.25 µl of the T4 DNA Ligase to the tube.
6. The total volume in each tube should now be 5 µl. Ensure the ligation is well-mixed by flicking the tube. You can spin the tube in a microcentrifuge for a few seconds to collect the liquid in the bottom of the tube again.

Incubations:

7. Incubate the reaction mix at room temperature for 30 min.
8. Incubate the reaction mix at 80C for 20 min. The 80C incubation deactivates the enzyme and improves transformation efficiency.
9. Store the ligation mix at -20C or proceed immediately to the transformation step.

# Assembly Protocol (Version 2)

Plasmid Collection:

1. Obtain kit plate and mark 24 wells containing
2. Pipette 1.5 uL of nuclease free water into specified wells
3. Pipette up and down
4. Collect all 24 well samples in a PCR tube

Plasmid Fast Digest

|  |  |
| --- | --- |
| Plasmid DNA | 1 ug (use the plasmid collection as an estimate) |
| 10X Thermo Scientific FastDigest Buffer | 2 uL |
| FastDigest Restriction Enzyme | 1uL |
| FastAP Thermosensitive Alkaline Phosphatase | 1uL |
| Water, nuclease-free | Up to 20uL |
| Total volume | 20 uL |

1. Mix thoroughly the reaction mixture above
2. Spin Briefly
3. Incubate at 37˚C for 10 minutes, stop by heating to 80˚C for 20 minutes

Insert DNA Restriction:

1. Remove the DNA and the destination plasmid along with NEBuffer 2 and BSA from the freezer to thaw. It is recommended to thaw the tubes by immersing them in room temperature water or remove them from the freezer and leave them in a cold box.

2. One PCR tube is for digest of the DNA part, and the last one is for the destination plasmid. Be sure to label each tube (DNA, P)
3. To each tube, add H2O and 100 ng of the DNA to be digested. Adjust the amount of water you add so the total volume in the DNA tube is 9.5 µl.
4. Add 1 µl of NEBuffer 2 to the DNA tube
5. Add 0.1 µl of BSA to the DNA tube.
6. Add 0.2 µl of the EcoRI to the DNA tube.

1. Only touch the very end of the pipette tip into the restriction enzyme!!!

7. Add 0.2 µl of the PstI to the DNA tube.
8. The total volume should be 10 µl for DNA. Ensure the digest is well-mixed by flicking the tube. You can spin the tube in a microcentrifuge for a few seconds to collect the liquid at the bottom of the tube again.
9. Return all reagents and purified DNA to the -20C freezer

Incubation:

10. Incubate the two restriction digests at 37C for 30 min. A water bath, an incubator, or a thermocycler are suitable for this incubation.
11. Incubate the two restriction digests at 80C for 20 min to deactivate the restriction enzymes. This step is most conveniently performed in a thermocycler so choose tubes for the restriction digest that fit in your thermocycler. As an easy way to confirm the digests worked, consider running 20 µl of each digest on a 1% agarose gel and look for bands of the expected length.
12. Store the two restriction digests at -20C or proceed immediately to the ligation step.

Ligation

|  |  |
| --- | --- |
| Linear Vector DNA | 20-100ng (6-10uL of Fast Digest) |
| Insert DNA | 2 Insert:1 linear vector molar ratio or about 12 uL |
| 10X T4 DNA Ligase Buffer | 2 uL |
| T4 DNA Ligase | 1 Weiss U = 1uL |
| Water, nuclease-free | Up to 20 uL |
| Total Volume | 20 uL |

1. Incubate 10 min at 22˚C
2. Use up to 5 uL of the mixture for transformation

# Transformation and Growth

Agar Plate Creation

1. Ratio of Agar to Water has to fall in a ratio equal to 10 grams of Agar to 400mL of tap water. Make sure to mix agar with water thoroughly before continuing with heating.
2. Heat up until the solution is golden-clear. Make sure to not over boil the agar.
3. Pour agar into 3 separate containers. Cool the medium-agar mix to 55°C.
4. Add antibiotics

|  |  |
| --- | --- |
| 2.5X | 25 ug/mL |
| 25X | 250 ug/mL |

5. Pour the plates. Use about 30 mL for each plate in a 100mm diameter plate
6. Allow the plates to set. If there are any bubbles in the plates, briefly pass the flame over to pop them.
7. Dry the plates in a 37°C incubator for 2-3 hours.
8. Use the plates immediately or seal them. Store the plates at 4°C

Transformation

1. Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins).
2. Remove [agar plates](https://www.addgene.org/protocols/pouring-lb-agar-plates/) (containing the appropriate [antibiotic](https://www.addgene.org/mol-bio-reference/antibiotics/)) from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator.
3. Mix 1 μl of DNA (usually 10 pg - 100 ng) into 20-50 μL of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times \*Pro-Tip\* Transformation efficiencies will be approximately 10-fold lower for [ligation](https://www.addgene.org/protocols/dna-ligation/) of inserts to vectors than for an intact control plasmid.
4. Incubate the competent cell/DNA mixture on ice for 20-30 mins.
5. Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 secs (45 secs is usually ideal, but this varies depending on the competent cells you are using).
6. Put the tubes back on ice for 2 min.
7. Add 250 μl LB or SOC media (without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min.
8. Plate some or all of the transformation onto a 10 cm [LB agar plate](https://www.addgene.org/protocols/pouring-lb-agar-plates/) containing the appropriate antibiotic.
9. Incubate plates at 37°C overnight

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# Team:CCA-San Diego/Results

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* Project

  [Description](https://2018.igem.org/Team%3ACCA-San_Diego/Description)
  [Design](https://2018.igem.org/Team%3ACCA-San_Diego/Design)
  [Parts](https://2018.igem.org/Team%3ACCA-San_Diego/Parts)
  [Results](https://2018.igem.org/Team%3ACCA-San_Diego/Results)
  [Applied Design](https://2018.igem.org/Team%3ACCA-San_Diego/Applied_Design)
  [Entrepreneurship](https://2018.igem.org/Team%3ACCA-San_Diego/Entrepreneurship)
* Experiments

  [Experiments](https://2018.igem.org/Team%3ACCA-San_Diego/Experiments)
  [Measurement](https://2018.igem.org/Team%3ACCA-San_Diego/Measurement)
  [Model](https://2018.igem.org/Team%3ACCA-San_Diego/Model)
  [Demonstrate](https://2018.igem.org/Team%3ACCA-San_Diego/Demonstrate)
  [Notebook](https://2018.igem.org/Team%3ACCA-San_Diego/Notebook)
  [Interlab](https://2018.igem.org/Team%3ACCA-San_Diego/Interlab)
  [Safety](https://2018.igem.org/Team%3ACCA-San_Diego/Safety)
* Parts

  [Parts](https://2018.igem.org/Team%3ACCA-San_Diego/Parts)
  [Basic Part](https://2018.igem.org/Team%3ACCA-San_Diego/Basic_Part)
  [Composite Part](https://2018.igem.org/Team%3ACCA-San_Diego/Composite_Part)
  [Part Collection](https://2018.igem.org/Team%3ACCA-San_Diego/Part_Collection)
  [Improve](https://2018.igem.org/Team%3ACCA-San_Diego/Improve)
* Human Practices

  [Human Practices](https://2018.igem.org/Team%3ACCA-San_Diego/Human_Practices)
  [Engagement](https://2018.igem.org/Team%3ACCA-San_Diego/Engagement)
* Team

  [Team](https://2018.igem.org/Team%3ACCA-San_Diego/Team)
  [Attributions](https://2018.igem.org/Team%3ACCA-San_Diego/Attributions)
  [Collaborations](https://2018.igem.org/Team%3ACCA-San_Diego/Collaborations)

# Results

.