As a part of our lab work, we’ve conducted numerous experiments with different protocols. The protocols we have used during our experiments were mostly based on the procedures of the companies which supplied us our genes and materials, including iGEM protocols. For example, for our Competent Cell Test, we’ve followed iGEM’s protocol; and for the plasmid isolation protocol, we’ve followed the protocol supplied by Nanobiz, our lab equipment provider.

Before starting each of our experiments, we have optimized and calculated the most suitable conditions. Below are the protocols we used as a result of our optimizations.

Resuspension of DNA in iGEM Kit Plates Protocol

Materials:

• dH2O
• iGEM Kit Plates
• Pipette

Method:

1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part desired.
2. Pipette 10 µLof dH2O into the well. Pipette up and down several times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. Resuspension will be in a crimson color, as the dried DNA has crisol dye.
3. Transform resuspended DNA into an eppendorf tube.

Competent Cell Preparation Protocol:

Buffer 1:

• Potassium acetate 30 µL
• RbCl 100 µL
• CaCl 100 µL
• 87% glycerol 4,3 mL
• Complete to 25 mL

Buffer 2:

• MOPS 10 µL
• RbCl 10 µL
• CaCl 75 µL
• 87% glycerol 4,3 mL
• Complete to 25 mL

Method:

1. Pipette 100 µL of grown KO11 into 10 mL LB with 2% glucose, incubate for 2 hrs at 37 ℃ till an OD value of 0,4-0,6 is reached.
2. Put 4,5 mL of cultures into falcon tubes, incubate on ice for 15 mins.
3. Centrifuge at 3500 RPM at 4 ℃ for 10 mins.
4. Discard supernatant.
5. Resuspend pellet on ice in ice-cold 650 µL of Buffer 1.
6. Centrifuge at 3500 RPM at 4 ℃ for 5 min.
7. Discard supernatant.
8. Resuspend pellet on ice in ice-cold 250 µL of Buffer 2.
9. Aliquot as 60 µL in 1,5 mL eppendorf.
10. Store at -80 ℃.

Overnight Culture in Falcon Tubes Protocol:

Materials:

• 50 mL Falcon tube
• Pipette and pipette tips
• Incubator
• Antibiotics
• Cultured bacteria
• LB broth

Method:

1. Put 10 mL LB broth in a 50 mL falcon tube.
2. Add 10 µL of antibiotics.
3. Get 100 µL of cultured bacteria and add to the falcon.
4. Place the tube in incubator for 16-18 hrs at 37℃, 135 RPM.

Competent Cell Test Kit Protocol

Materials:

• 70% ethanol
• Paper towels
• Lab marker / Sharpie
• 1,5 mL microcentrifuge tubes
• Container for ice
• Ice
• Competent cell aliquot(s)
• Competent Cell Test Kit
• Agar plates with chloramphenicol
• 42°C Water Bath (or hot water source and thermometer)
• 37°C Incubators (oven and shaker)
• LB
• Sterile glass beads or sterile cell spreader
• Pipette
• Pipette tips

Method:

1. Clean your working area by wiping down with 70% ethanol.
2. Thaw competent cells on ice. Label 1,5 mL microcentrifuge tubes for each transformation and then pre-chill by placing the tubes on ice.
3. Spin down the DNA tubes from the Competent Cell Test Kit/Transformation Efficiency Kit to collect all of the DNA into the bottom of each tube prior to use. A quick spin of 20-30 seconds at 8,000-10,000 RPM will be sufficient. Note: You should resuspend the DNA in each tube with 50 µL of dH2O.
4. Pipet 1 µL of DNA into each microcentrifuge tube.
5. Pipet 50 µL of competent cells into each tube. Flick the tube gently with your finger to mix.
6. Incubate on ice for 30 minutes.
7. Pre-heat water bath now to 42°C. Otherwise, hot water and an accurate thermometer works too!
8. Heat-shock the cells by placing into the water bath for 45 seconds. Be careful to keep the lids of the tubes above the water level, and keep the ice close by.
9. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes.
10. Add 950 µL of LB media per tube, and incubate at 37°C for 1 hour shaking at 200-300 RPM.

Prepare the agar plates during this time: label them and add sterile glass beads if using beads to spread the mixture.

11. Pipet 100 µL from each tube onto the appropriate plate, and spread the mixture evenly across the plate. Incubate at 37°C overnight or approximately 16 hours. Position the plates with the agar side at the top, and the lid at the bottom.
12. Count the number of colonies on a light field or a dark background, such as a lab bench. Use the following equation to calculate your competent cell efficiency. If you've done triplicates of each sample, use the average cell colony count in the calculation. Efficiency (in cfu/µg) = [colonies on plate (cfu) / Amount of DNA plated (ng)] x 1000 (ng/µg) Note: The measurement "Amount of DNA plated" refers to how much DNA was plated onto each agar plate, not the total amount of DNA used per transformation. You can calculate this number using the following equation: Amount of DNA plated (ng) = Volume DNA added (1 µL) x concentration of DNA (refer to vial, convert to ng/µL) x [volume plated (100 µL) / total reaction volume (1000 µL)]

Overnight Culture in Falcon Tubes Protocol:

gBlocks come as 1000 ng (basic 1 and basic 2)

Method:

1. Centrifuge the tube for 3-5 seconds at a minimum of 3000 G to ensure the material is in the bottom of the tube.
2. Add water to reach a final concentration of 20 ng/µL (Add 50 µL of water).
3. Vortex briefly.
4. Incubate at 50 ℃ for 20 min.
5. Briefly vortex and centrifuge.

Seed Stock Protocol:

Materials:

• Sterile pipette
• LB (containing 2% glucose
• 5 µL chloramphenicol
• Sterile glassware and plasticware
• Incubator
• Falcons

Method:

1. Take 5 mL of LB.
2. Add 5 µL of chloramphenicol (40 mg/L).
3. Take 0,5 mL of LB+CHL with a pipette, dissolve bacteria with LB.
4. Pour back into the 5 mL medium

80% Glycerol Stock Protocol:

Materials:

• 80% glycerol solution (it can be obtained by mixing 80 mL pure glycerol with 20 mL distilled water and autoclaving)
• Desired bacterial culture
• Microcentrifuge tubes
• Pipettes and pipette tips

Method:

1. Using a sterile pipette tip, transfer 0,75 mL of 80% glycerol in each of the eppendorf tubes.
2. After adding the glycerol to the tubes, add 0,75 mL of cultured bacteria.
3. Mix by pipetting up and down.
4. When done, place them in -80°C freezer.

Digestion Protocol:

Materials:

• Enzyme Mastermix for plasmid backbone (25µLtotal, for 5 rxns.)
• 5 µL NEB Buffer 2 (vortex)
• 0,5 µL EcoR1-HF
• 0,5 µL Pst1
• 19 µL dH2O
• Digest plasmid backbone (pSB1C3)

Method:

1. Add 4 µL of linearized plasmid backbone (25 ng/µLfor 100 ng total).
2. Add 4 µL enzyme Mastermix.
3. Digest 37 ℃/12 hrs, heat kill 80 ℃/20 min.

Restriction Digestion Protocol

Materials:

• Biobrick part in Biobrick plasmid
• dH2O
• NEBuffer 2
• EcoR1
• Pst1
• Ice

Method:

1. Add 250 ng of DNA (12,5 µL+ 3,5 µL water) to be digested, and adjust with dH2O for a total volume of 16 µL.
2. Add 2,5 µL of NEBuffer 2.
3. Add 0,5 µL of EcoR1.
4. Add 0,5 µL of Pst1.
5. Incubate at 37 ℃ for 12 hrs. 80 ℃/20 min heat kill the enzymes.
6. iRun a portion of the digest on a gel (8 µL, 100 ng) to check all are accurate.

LB Agar Preperation

Materials:

• 5 gr bacto-tryptone
• 2,5 gr yeast extract
• 5 gr NaCl
• 7,5 gr agar
• Distilled water up to 500 mL

Method:

1. Put all materials in a 500 mL lab bottle.
2. Autoclave.

LB Broth Preperation:

Materials:

• 5 gr bacto-tryptone
• 2,5 gr yeast extract
• 5 gr NaCl
• Distilled water up to 500 mL

Method:

1. Put all materials in a 500 mL lab bottle.
2. Autoclave.

Transformation Protocol:

Materials:

• DNA
• competent cells
• 1,5 mL microtubes
• LB broth
• Petri plates/ LB agar/ antibiotic (2 per sample)

Equipment:

• Floating Foam Tube Rack
• Ice & ice bucket
• Lab Timer
• 42°C water bath
• 37°C incubator
• Sterile spreader or glass beads
• Pipettes and Tips
• Microcentrifuge

Method:

1. Resuspend DNA.
2. Label 1,5 mL tubes with part name. Fill lab ice bucket with ice, and pre-chill 1.5 mL tubes.
3. Thaw competent cells on ice.
4. Pipette 50 µLof competent cells into 1,5 mL tube.
5. Pipette 1 µLof resuspended DNA into 1,5 mL tube.
6. Close 1,5 mL tubes, incubate on ice for 30 mins.
7. Heat shock tubes at 42°C for 45 sec.
8. Incubate on ice for 5 mins.
9. Pipette 950 µL LB media to each transformation.
10. Incubate at 37°C for 1 hours, shaking at 200-300 RPM.
11. Pipette 100 µL of each transformation onto petri plates.
12. Incubate transformations overnight (14-18 hr) at 37°C.
13. Pick single colonies.

Ligation:

Materials:

• T4 DNA ligase buffer (10X)
• T4 DNA ligase
• Vector DNA
• Insert DNA
• (Complete the solution to 10 µL with water) Nuclease-free water.

The proportions we used for our parts are below:

	basic 1 (1:3 ratio)	basic 2 (1:3 ratio)	composite 1 (1:3 ratio)	composite 2(1:2 ratio)	composite 3 (1:1,5 ratio)
T4 DNA ligase buffer (10X)	1 µL	1 µL	1 µL	1 µL	1 µL
Vector DNA	2 µL	2 µL	2 µL	2 µL	2 µL
Insert DNA	4 µL	4,85 µL	3,34 µL	4,38 µL	5,3 µL
Nuclease-free water	2,5 µL	1,65 µL	3,16 µL	2,12 µL	1,2 µL
T4 DNA ligase	0,5 µL	0,5 µL	0,5 µL	0,5 µL	0,5 µL

Method:

Place all materials into PCR tubes in the order of

1. Water
2. T4 DNA ligase buffer
3. Vector DNA
4. Insert DNA
5. T4 DNA ligase

Total of 2 tubes and then into the PCR. (overnight 16°C, heat kill 65°C)

Plasmid Isolation protocol, kit from Nanobiz

1. Add 2 mL of bacteria to an eppendorf tube and at maximum speed centrifuge for 2 min.
2. Discard supernatant.
3. Add buffer P1, 200 µL to pellet, mix Add buffer P2, 200 µL, mix well until blurry image is gone. Add buffer P3, 200 µL, mix well.
4. 5 min centrifuge, discard pellet this time.
5. About 400 µL of supernatant should be obtained.
6. Transfer to sterile microcentrifuge tube.
7. Add 600 µL of buffer P4, mix well.
8. Transfer to spin colon (600 µL) and centrifuge at 10.000 RPM for 1 min. (Discard collection tube liquid) Add remaining 400 µL, 10.000 RPM 1 min centrifuge. (Discard collection tube liquid).
9. Add 500 µL of buffer P5.
10. 10.000 RPM 1 min centrifuge. Discard the liquid at the bottom of the collection tube.
11. 2 min centrifuge 14.000 rpm.
12. Discard the liquid at the bottom of the collection tube.
13. Transfer to a suitable eppendorf (1,5 mL tubes).
14. Pour 30-50 µLwater carefully (We added 40 µL).
15. Wait for 2 min at room temperature.
16. 1 min 6000 RPM centrifuge.
17. Run on gel.

Gel Preparation and Loading Protocol:

Materials:

• 1X TAE 50cc
• 0,75 gram Agarose
• 2,5 µL EtBr

*Materials are multiplied by 3 if done with a large bath.

Method:

1. Mix 1 µL of 6X Loading Dye and 5 µL of PCR Product per sample by pipetting up and down on a parafilm sheet.
2. Load on wells starting from the second well.
3. Load 6 µL of ladder on first and last wells.
4. Run on gel for 30 min with a voltage of 90.

Colony PCR Protocol

Mastermix Materials:	25 µL (1X)
10X Taq Buffer	2,5 µL
25mM MgCl2	2,5 µL
2 mM dNTP	2,5 µL
10uM Primer F	2,5 µL
10uM Primer R	2,5 µL
dH2O	11,4 µL
Taq DNA Polymerase	0,125 µL

1. Label eppendorfs.
2. Put 30 µL dH2O to each eppendorf.
3. Get PCR tubes and label.
4. Choose single colonies from overnight cultures.
5. Get empty plates and divide according to the number of single colonies chosen.
6. Get half of single colonies with a pipette tip, mix well in the labeled eppendorf tubes and then inoculate on plates accordingly.
7. Centrifuge the eppendorfs and plate in water bath in 95 ℃ for 10 minutes. Prepare mastermix while eppendorf tubes are in water bath.
8. Centrifuge the eppendorfs at 13.000 RPM for 5 min.
9. Distribute 23 µL of Mastermix to PCR tubes.
10. Add 2 µL of template.
11. Place the PCR tubes in PCR; plates in incubator.

    initial denaturation	95 ℃	5 min
    extension (30 cycle)	95 ℃	30 sec
    extension (30 cycle)	60 ℃	30 sec
    extension (30 cycle)	72 ℃	1 min
    final extension	72 ℃	5 min
    hold	4 ℃	∞

12. Load and run on gel.

PCR Protocol:

Mastermix Materials:	25 µL (1X)
Taq Buffer	2,5 µL
25 mM MgCl2	2,5 µL
2 mM dNTP	2,5 µL
10 µM Primer F	2,5 µL
10 µM Primer R	2,5 µL
dH2O	11,4 µL
Taq DNA Polymerase	0,125 µL

1. Label PCR tubes.
2. Prepare master-mix and distribute to PCR tubes.
3. Add template.
4. Place in PCR.

   initial denaturation	95 ℃	5 min
   extension (30 cycle)	95 ℃	30 sec
   extension (30 cycle)	60 ℃	30 sec
   extension (30 cycle)	72 ℃	1 min
   final extension	72 ℃	5 min
   hold	4 ℃	∞

5. Load and run on gel.

Interlab Protocol:

iGEM protocol is followed, link is provided below:
https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf
iGEM protocol is followed, link is provided below:
http://parts.igem.org/Help:Submission_Kit#Submission_DNA_Preparation