Materials
- Chemically competent cell
- 250μL of SOC or LB media
- Plates with corresponding antibiotic for selection

Step by step
1. In an eppendorf tube, mix 1ng of plasmid DNA with 50μL of chemically competent cell on ice. Don’t leave the competent cells on ice for more than 10 minutes.
2. Flick them very softly and leave them on ice for 30 min.
3. Heat-shock them at 42°C for exactly 45 seconds.
5. Add 250μL of SOC or LB media into the tube.
6. Leave the bacteria at 37ºC for an hour.
7. Spin the cells for a minute or until pellet is visible.
8. Discard 200μL of supernatant
9. Resuspend the pellet in the remaining liquid and plate 50μL in a petri dish with the corresponding antibiotic.

Materials
- SOB
- 1 M filter sterilized glucose
Step by step
1. Add to 98 mL of SOB, 2mL of the glucose stock solution.

Materials
- Yeast extract
- Tryptone
- NaCl
- KCl
- MgSO4

Step by step
1. Mix in a flask per 500mL:

2.5 g Yeast extract
10 g Tryptone
0.292 g NaCl
0.093 g KCl
1.2 g MgSO4
500 ml of dH20
2. Adjust to pH 7.5 prior to use. This requires approximately 12.5 ml of 1M NaOH.
3. Send to autoclave.

Materials
- LB broth
- dH2O
Step by step
1. Mix in a flask:

25g of LB broth
Required H20
1L Total

2. Dissolve completely.
3. Autoclave.

Materials
- LB broth
- Agar
- dH2O

Step by step
1. Mix in a flask:

25g of LB broth
15 g of agar
Required H20
1L Total

2. Dissolve completely (you can heat it in the microwave in order to do so).
3. Autoclave.

1. Use the same protocol as E. coli transformation but change step 1.

New step 1:
In an eppendorf tube, mix 50 ng of each plasmid DNA with 50μL of chemically competent cell on ice.

Materials
- 5mL of overnight culture
- Plasmid DNA isolation miniprep kit 

Step by step
1. Inoculate 5mL of liquid LB with the corresponding antibiotics with the bacteria that has the plasmid of interest. Use a pipette tip to grab a single colony from a plate or inoculate 1μL from a glycerol stock.
2. Leave the liquid cultures in a rotating shaker at 37ºC for the night.
3. Spin at 3000 rpm for 10 minutes.
4. Discard supernatant.
5. Follow manufacturer’s instructions from the Miniprep DNA Purification System that you have.

Our team used:
Promega’s miniprep kit and centrifugation protocol
For a better concentration, do the final elution step with the minimum volume possible specified in the kit’s manual.

Materials
- Agarose gel band
- Gel and PCR Clean-Up System kit

Before gel purification:
1. Separate your DNA fragments using an agarose gel electrophoresis.
2. Cut the DNA band with the expected size out of gel using UV light at 70% power. Cut the band with a scalpel. To reduce DNA damage, try to minimize as much as possible the gel exposure to UV light.

Step by step
3. Write down the weight of your gel piece.
4. Follow manufacturer’s instructions from the Gel and PCR Clean-Up System kit that you have.

Our team used:
Promega’s Wizard® SV and PCR Clean-Up System and centrifugation protocol
For better concentration, do the final elution step with the minimum volume possible specified in the kit’s manual.

Materials
- Purified plasmid of interest
- Nuclease free water
- BSA 100X
- Restriction enzyme
- Adequate buffer for the restriction enzyme (or combination of enzymes) that you want to use *.

Step by step
1. Mix on ice in a PCR tube:

a. For a single restriction digest reaction:

X volume of plasmid to reach 300-500ng DNA
(43.5 - X)μL of nuclease free water
5μL of adequate 10X buffer
0.5μL of BSA 100X
1μL of Restriction enzyme (add last)
50μL total

b. For a double restriction digest reaction:

X volume of plasmid to reach 300-500ng DNA
(42.5 – X)μL of nuclease free water
5μL of 10X buffer, select according to the enzyme
0.5μL of BSA 100X
1μL of Enzyme 1
1μL of Enzyme 2
50μL total

2. Incubate the mix at 37ºC for about 2 hours. It depends on the size of the plasmid.
3. Use 20μL of the reaction to run an agarose gel of electrophoresis. Add loading dye.
* You can usually find the correct buffer in your supplier’s webpage. In our case: New England Biolabs (LINK TO: https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder)

Materials
- DNA template
- Nuclease free water
- Master Mix 2X
- Primer Forward
- Primer Reverse

Step by step
1. Mix on ice in a PCR tube:

1ng of Template DNA
0.75μL of Primer forward 10 μM
0.75μL of Primer Reverse 10 μM
15μL of Master Mix 2X*
12.5μL of Nuclease free water
30μL Total

2. Put the tube in the thermocycler and select the cycling conditions.
Depending on complexity of template DNA (GC content or highly similar sequences), presence of spurious products, or enhance the final yield, different cycling conditions can be used on the PCR.
a. Normal PCR
1. Initialization: 30 seconds at 98 ºC
2. Denaturation: 10 seconds at 98 ºC
3. Annealing: 30 seconds at 56-61 ºC (depending on primer melting temperatures)
4. Elongation: 30 seconds per Kb to amplify at 72 ºC
Steps 2 to 4 are repeated from 30 to 35 times.
5. Final extension: 7 minutes at 72 ºC

b. Touchdown PCR
Touchdown PCR uses higher temperatures for the annealing of the primer with the template for more specific pairing to avoid nonspecific amplifications.

1. Initialization: 30 seconds at 98 ºC
2. Denaturation: 10 seconds at 98 ºC
3. Annealing: 30 seconds at 72 ºC decreasing 1 ºC in each cycle
4. Elongation: 30 seconds per Kb to amplify at 72 ºC
Steps 2 to 4 are repeated 17 times.
5. Denaturation: 10 seconds at 98 ºC
6. Annealing: 30 seconds at 54ºC
7. Elongation: 30 seconds per Kb at 72 ºC
Steps 5 to 7 are repeated 15 times.
8. Final extension: 7 min at 72 ºC

c. Hot Start Touchdown PCR
The idea is for the reaction to have a longer denaturation time so the primers can anneal with a higher probability to the template than with themselves.

Materials
- Master Mix (MM) 2X (Either HF, GC or GC+DMSO) without Phusion Polymerase
- Template DNA
- Primer Forward
- Primer Reverse

Step by step
1. Make your MM 2X according to the instructions above but without adding the polymerase.
2. Prepare a dilution of the Phusion polymerase according to the concentration needed in each tube.
3. In a PCR tube, mix the MM 2X with the primers and the template DNA (same as above).
4. Put the tubes in the thermocycle with the following cycling conditions:

a. Initialization: 30 seconds at 98 ºC
b. Denaturation: 4 minutes at 98 ºC
c. Annealing: 30 seconds at 72 ºC decreasing 1 ºC in each cycle
d. Elongation: 30 seconds per Kb to amplify at 72 ºC
Steps 2 to 4 are repeated 17 times.
e. Denaturation: 10 seconds at 98 ºC
f. Annealing: 30 seconds at 54ºC
g. Elongation: 30 seconds per Kb at 72 ºC
Steps 5 to 7 are repeated 15 times.
h. Final extension: 7 min at 72 ºC

4. While on the first denaturation step (b), open the PCR machine and quickly add the dilution of the polymerase to the tube.
5. Close the thermocycle and let it continue as normal.

2X MM HF buffer:
1. Mix on ice in a 1.5 mL eppendorf tube:

Buffer 5X HF 200μL
dNTPs 20μL
Phusion Poly 2U/μL 10μL
Nuclease free Water 270μL
500μL Total

2. Make aliquot of 15μL of solution in PCR tubes
3. Store at -20°C.
2X MM GC buffer
1. Mix on ice in a 1.5 mL eppendorf tube:

Buffer 5X GC 200μL
dNTPs 20μL
Phusion Poly 2U/μL 10μL
Nuclease free Water 270Μl
500μL Total

2. Make aliquot of 15μL of solution in PCR tubes
3. Store at -20°C.

2X MM GC+DMSO buffer (if primer dimers were observed in previous PCR attempts):
1. Mix on ice in a 1.5mL eppendorf tube:

Buffer 5X HF 200μL
dNTPs 20μL
Phusion Poly 2U/μL 10μL
DMSO 5% 50μL
Nuclease free Water 220μL
500μL Total

2. Make aliquot of 15μL of solution in PCR tubes
3. Store at -20°C.

Materials
- Master Mix 2X GoTaq Flexi
- Plate or liquid culture of the bacteria

Step by step
1. Mix on ice in a PCR tube:
Template DNA: Either grab 1μL of the liquid culture or pick a single colony with a micropipette point and deposit it at the bottom of a PCR tube.
0.75μL of Primer forward, 10 μM
0.75μL of Primer Reverse, 10 μM
15μL of Master Mix 2X GoTaq Flexi
12.5μL of Nuclease free water
30μL Total

2. Put the tube in the thermocycler and select the cycling conditions.
1. Initialization: 10 minutes at 95ºC (longer initialization step for bacterial breakage)
2. Denaturation: 30 seconds at 95 ºC
3. Annealing: 30 seconds at 56-61 ºC (depending on primer melting temperatures)
4. Elongation: 30 seconds per Kb to amplify at 72 ºC
Steps 2 to 4 are repeated from 30 to 35 times.
5. Final Extension: 7 minutes at 72 ºC

MM 2X GoTaq Flexi:
1. Mix in a 1.5 mL eppendorf tube:

5X GoTaq Buffer 200μL
MgCl2 50mM 50μL
dNTPs 10mM 20μL
GoTaq Flexi Polymerase 6μL
Nuclease free water 224μL
500μL Total

2. Make aliquot of 15μL of solution in PCR tubes
3. Store at -20°C.

Materials
- TAE 1X buffer
- Agarose
- GelRed 10000X Nucleic Acid Stain
- Loading dye 6X
- DNA sample

Step by step
1. Prepare gel by adding agarose to TAE 1X buffer to a final concentration of 1-3%.
2. Heat the mixture until it is fully dissolved and looks transparent.
3. Let it stand until it is warm and then add the RedGel to a final concentration of 1000X.
4. Set up your gel. Add a comb, this depends on the width you want your wells to be and the number of samples to load.
5. Wait until the gel solidified.
6. Prepare your samples by adding DNA loading dye 6X to a final concentration of 1X.
7. Load them into the gel before submersion.
8. Run the electrophoresis at 80-120V for about 40 minutes in TAE 1X buffer.
9. Reveal the DNA bands using an UV transilluminator.

50x TAE:
For 1 L
1. Dissolve 242.2 g Tris base in around 600 mL of ddH2O
2. Slowly add 57.1 mL glacial acetic acid
3. Add 100 mL 0.5 M EDTA, pH 8
4. Bring up the volume to 1 L with ddH2O

6x DNA loading buffer:
For 100 mL
1. Weigh 60 g glycerol into a 100-mL graduated cylinder
2. Add 12 mL 0.5 M EDTA, pH 8
3. Add 10 mg bromophenol blue
4. Bring up the volume to 100 mL with ddH2O

Materials
- Gibson Assembly master mix 1.33X
- Amplified DNA fragments

Step by Step
1. Mix on ice in a PCR tube:

Assembly reaction contains:
9μL of 1.33X Gibson assembly master mix
3μL of purified parts DNA*
12μL Total

2. Incubate at 50ºC for 1 hour.
3. Transform chemically competent cells with 12μL of assembled DNA.

* Ratio of parts to vector backbone should be 3:1 in weight, except when part is smaller than 200 bp. In that case, ratio should be 5:1.

Gibson Assembly Master Mix 1.33X preparation
1. Mix on ice:

100μL 5X Gibson Assembly Isothermal Buffer
6.25μL Phusion Polymerase 2 U/μL
2μL T5 Exonuclease 1U/μL
50μL Taq DNA Ligase 2000 U/μL
216.75μL nuclease free H2O.
500 μL Total (for approximately 41 reactions)

2. Make aliquot of 9μL of solution in PCR tubes.
3. Store at -20°C.

We recommend using a protocol found in openwetware
Materials
- All glassware needs to be autoclaved with ddH2O prior to the protocol to remove any remaining detergent
- SOB
- CCMB80 buffer

Step by step
1. Inoculate a single colony of TOP10 E. coli into 250 mL of SOB media in a 500 mL Erlenmeyer flask.
2. Incubate at 37ºC in a rotating shaker until OD600 of 0.5 is reached.
3. Collect the bacteria in five 50mL sterile falcon tubes and centrifuge at 4200 rpm at 4°C for 10 minutes.
4. Discard supernatant.
5. Resuspend the cells with 20 mL of CCMB80 buffer by pipetting.
6. Incubate the suspension on ice for 20 minutes.
7. Centrifuge at 4200 rpm at 4°C for 10 minutes.
8. Discard supernatant.
9. Resuspend the cells with 2mL of CCMB80 buffer in each Falcon tube by pipetting.
10. Test OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells.
11. Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
12. Make aliquots in ice chilled 1.5mL Eppendorf tubes and store at -80ºC.
13. Test the efficiency of your competent; you should obtain competence levels of 5*10⁸ cfu/μg of pUC19 DNA.

CCMB80 buffer
Materials
- 1M KOAc, pH 7.0
- CaCl2.2H2O
- MnCl2.4H2O
- MgCl2.6H2O
- 10% glycerol (100 ml/L)
- 0.1N HCl

Step by step
1. Mix in a flask:

10 mL of 1M KOAc, pH 7.0
11.8 g/L CaCl2.2H2O
4.0 g/L MnCl2.4H2O
2.0 g/L MgCl2.6H2O
100 mL/L of 10% glycerol
1L Total

2. Adjust pH to 6.4 with 0.1N HCl
3. Sterile filter
4. Store at 4°C

Materials
- L-arabinose (1M)
- IPTG (0.1 M)
- Liquid culture of co-transformed bacteria

Step by step
1. Inoculate 5mL of liquid LB with the corresponding antibiotics the bacteria that has the co-transformed plasmids of interest. Use a pipette tip to grab a single colony from a plate or inoculate 1μL from a glycerol stock.
2. Leave the liquid cultures in a rotating shaker at 37ºC for the night.
3. Early in the morning check out the OD of the culture using a spectrophotometer.
4. The OD needs to be approximately 0.5 (600nm). Adjust by alternating between making dilutions and leaving the culture on the 37ºC shaker.
5. Add 50μL of L-Arabinose and 2.53μL of IPTG to the bacteria culture.
6. Wait for around 4 hours.
7. Watch GFP activity on the microscope.

Materials
- Xilol
- Lens Cleaning Tissue
- Microscope slides
- Epifluorescence microscope. Ours was Nikon Eclipse Ni

Step by step
A. Sample preparation:
1. Centrifuge bacterial flask at 3.000 rpm for 10 minutes at 25ºC
2. Discard supernatant
3. Resuspend bacteria with the medium left in the flask
4. Place 20 μL of the sample on a glass slide
5. Place a cover slip on top of the sample

B. Observation:
For the observation under the microscope, the first step is to begin with the lowest magnification. Focus the sample and from there, progressively amplify it using the different objective lens.
When using the maximum amplification (100x objective lens) the use of immersion oil it is needed. Just apply it between the cover slip and the objective lens (until the lens is touching it).

Remember after finish to clean up the objective with Xilol.

Starting with the 20x objective lens (or any lower) may become difficult if you are not using a staining method, because the magnification is not sufficient for the identification of single bacteria. A good trick is using any particle or contamination that may be within the sample to help with the focus.
The observation of fluorescence is very similar regarding the focusing of the sample but since it uses an excitatory wavelength, it’s necessary to turn off the light and use a special filter depending on the type of fluorescence used.

B. Image Acquisition
All the images are obtained with a camera coupled to the microscope and recorded on a computer using special software. We used NIS-Elements software.

Materials
- Sterile 80% glycerol
- Sterile cryogenic storage vials
- Liquid culture of the bacteria to be stored

Step by step
1. In the sterile tube, add 500 μL of 80% glycerol with 500 μL of the liquid culture.
2. Thoroughly mix by inverting.
3. Wrap a piece of parafilm around the lid.
4. Store at -80ºC.

Ampicillin (1000x):
• Dissolve 5 g ampicillin in 25 mL ddH2O.
• Add 25 mL absolute ethanol.
• Store at -20 °C.

Chloramphenicol (1000x):
• Dissolve 1.7 g chloramphenicol in 50 mL absolute ethanol.
• Store at -20 °C.

Kanamycin (1000x):
• Dissolve 1.25 g ampicillin in 50 mL ddH2O.
• Sterilize by filtering through 0.2 μ.
• Store at -20 °C in 1.5-mL aliquots.
*Note: for autoinduction media a 250x of Kanamycin is used.

Tetracycline (1000x):
• Dissolve 0.75 g tetracycline in 50 mL absolute ethanol or isopropanol.
• Store at -20 °C in 1.5-mL aliquots.

Materials
- Agar LB
- Petri dishes
- Stock of selection(s) of bacteria of interest

Step by step
(For 1 plate)

1. Microwave around 30 mL of LB agar until fully melted.
2. Wait until warm.
3. Add appropriate amount of antibiotic or selection of bacteria of interest*.
4. Spread it in a petri dish (careful not to leave any bubbles).
5. Close the lid when dry.

* Using C1*V1=C2*V2
Where
C1 = concentration of the stock
V1= Volume you need to add
C2= Final concentration
V2= Final volume

Materials
- Nuclease free water
- 5x Green Buffer
- DNA Ladder 1K

Step by step
1. Mix on ice:

700 μL of H2O
200 μL of 5x Green Buffer
100 μL of DNA Ladder 1K
1 mL total

Materials
- TEMB buffer
- CelLytic B-II (Sigma)
- M-280 streptavidin-coated Dynabeads
- MiniMACS magnet

Step by step
Carboxysome purification
1. Grew 3 liters of induced bacteria.
2. Centrifuge at 3.000 RPM for 10 minutes at 25ºC to obtain bacterial pellet.
3. Resupend on 50mL of TEMB buffer.
4. Sonication of cell suspension in an icebath. 4 times, 30 seconds each.
5. High speed centrifugation at 12.000 x g for 20 minutes.
6. Collect the supernatant and resuspend in 20mL of a 30% (vol/vol) solution of CelLytic B-II in TEMB.
7. Centrifuge at 40.000 x g for 30 minutes. After this you should obtain a carboxysome-enriched pellet.
8. Resuspend in 3mL of TEMB buffer.

Carboxysome extraction
1. Place the needed amount of M-280 streptavidin-coated Dynabeads (25 μl or ~1.5 × 107 beads per purification) into a 1.5-ml tube, then add 1 ml of resuspended carboxysomes and invert the tube several times to wash the beads. Pellet the beads by centrifugation at 3,500g for 2 min at 4 °C. Decant and discard the supernatant and resuspend the beads in their original volume with TEMB buffer.
2. Add 25 μl of the bead slurry from Step 1 to the carboxysome suspension. Place the tube on a rotating mixer and rotate at 4 °C for 30 min. After the 30-minutes incubation, remove 20 μl of this solution for analysis of carboxysomes preparation and bead binding through epifluorescence microscopy.
3. Transfer the 1mL mixture of carboxysome and beads from Step 2 into a 15mL tube. Gently add 9mL ice-cold TEMB buffer to the 1mL of carboxysome/bead suspension. Invert gently to mix and keep this suspension at 4 °C while performing Steps 4–6.
4. Begin setting up the flow purification column in the 4 °C cold room. With the MiniMACS magnet oriented horizontally on the bench, slide a TEMB-filled 1-ml pipette tip through the groove in the magnet until the narrow end of the tip protrudes 2.5 cm from the other side of the magnet. The part of the pipette tip running through the groove should be in full contact with the surface of the magnet. All subsequent steps of the carboxysome purification are also performed at 4 °C.
5. Place the magnet with inserted 1mL tip into the ring stand clamp so that the tip is vertical, and allow the TEMB solution in the tip to drain into a beaker. Attach a stopcock to the narrow end of the tip by a 2-cm length of tubing.
6. Close the stopcock and fill the 1mL pipette tip with TEMB until the level of liquid is 1 cm from the top of the tip. 7. Gently invert the Carboxysome/bead mixture from Step 3 several times to mix, and then draw the solution into a 10mL serological pipette using an electronic serological pipetting device.
8. With the 10mL pipette still attached to the pipetting device, gently but firmly secure the narrow end of the 10mL pipette into the wide end of the 1mL pipette tip in the magnet. Carefully remove the pipetting device from the top of the 10mL pipette. The flow purification device is now completely assembled.
9. Gradually open the stopcock on the purification device until a flow rate of 0.75mL min− 1 is reached. Allow the column to drain completely, with the flowthrough draining into a beaker. A collection of beads should become visible on the wall of the 1mL tip as the liquid flows past.
10. Once all liquid has drained from the column, including the 1mL pipette tip, disassemble the column by removing the 10mL pipette and tubing from the ends of the 1mL pipette tip, and then gently removing the 1mL tip from the groove of the magnet.
11. Place 1mL ice-cold TEMB into a 1.5mL tube. Insert the 1mL pipette tip containing collected beads and nuclei onto a micropipette and use it to gently and repeatedly draw the 1mL TEMB into and out of the tip until all beads are released into solution.
12. Using the 1mL of carboxysome/bead mixture from Step 3, perform a second round of flow purification by repeating Steps 3–11. Remove 20 μl from the 1mL of final purified nuclei suspension for analysis of purity, same as in step 2.
13. Centrifuge the 1mL purified carboxysome/bead mixture at 1,000 x g for 5 min at 4 °C to collect bead-bound carboxysome. Carefully decant the supernatant and resuspend the pellet in 20 μl ice-cold TEMB and place on ice.
14. Store purified carboxysomes at -80ºC.