Team:UPO-Sevilla/Protocols

PROTOCOLS

  1. Collect the saturated bacteria culture in a 1'5 mL tube and centrifuge at 13000 rpm for 1 minute. Do it twice

  2. Add 100 μL of GTE (Tris HCl 25 mM, pH 8, glucose 50 mM, EDTA 10 mM), mix by inverting and incubate on ice for 5 minutes

  3. Add 150 μL of potassium acetate 3 M (pH 4'8), mix by inverting and incubate on ice for at least 5 minutes

  4. Centrifuge the tubes at 13000 rpm for 10 minutes and transfer the supernatant to a 1'5 mL tube

  5. Add 400 μL of ice ethanol 96% and centrifuge at 13000 rpm for 10 minutes

  6. Discard the supernatant, add 500 μL of ethanol 70% and centrifuge at 13000 rpm for 5 minutes

  7. Let the tubes dry and resuspend the plasmid DNA in 50 μL of TER (TrisHCl 10 mM, pH 8, EDTA 1 mM, RNAse 20 mg L -1)

GEL MAKING

  1. Prepare 40 or 100 mL of TAE (Trisacetate 40 mM, pH 7’7, EDTA 10 mM), it depends on the size of the gel

  2. Add 0’4 g or 1 g of agarose to the TAE solution (for 1% gel)

  3. Heat the solution until all the agarose has been dissolved and cold it to 50ºC

  4. Pour the the solution in the electrophoresis vessel, apply the combs and let it polymerize

GEL RUNNING

  1. Add 1 μL of loading buffer 6x (TrisHCl 5 mM, pH 8, EDTA 0’5 mM, glycerol 30%, xylene cyanol 2’5 g L -1, bromophenol blue 2’5 g L -1) to 5 μL of DNA sample

  2. Remove the combs from the gel, put the vessel on a horizontal electrophoresis system and cover it with TAE

  3. Pipette 6 μL of the DNA samples and ladder

  4. Run at 120 mV for 45 minutes approximately

REVEAL THE GEL

  1. Put the gel in ethidium bromide for 20 minutes

  2. Look the gel on the transilluminator and take a photo of it

  1. Add 6 μL of insert DNA

  2. Add 1 μL of vector DNA

  3. Add 2 μL of ligase buffer high ATP (TrisHCl 50 mM, pH 7’6, MgCl 2 10 mM, DTT 5 mM, BSA 50 μg mL -1)

  4. Add 1 μL of T4 DNA ligase

  5. Incubate at 16ºC overnight

  1. Plate Escherichia coli DH5α and incubate at 37ºC overnight

  2. Pick a colony, inoculate in LB media and incubate at 37ºC and 180 rpm overnight

  3. Dilute from 100 to 500 times the culture in SOB media(tryptone 20 g L-1, extract of yeast 5 g L-1, NaCl 8’5 mM, KCl 1’25 mM, MgCl2 10 mM) and incubate at 20ºC until a DO600=0’6

  4. Cold the culture on ice for 10 minutes and collect the cells by centrifuging at 4ºC and 3000 g for 10 minutes

  5. Discard the supernatant, resuspend in 80 mL of iced TB (PIPES 10 mM, CaCl 2 15 mM, KCl 250 mM, MnCl2 55 mM, pH 6’7) and incubate on ice for 10 minutes

  6. Centrifuge at 4ºC and 3000 g for 10 minutes, discard the supernatant and resuspend in 20 mL of TB

  7. Add DMSO (dimethyl sulfoxide) until a final concentration of 7% (v/v) and incubate on ice for 10 minutes

  8. Distribute the cells to samples of 100 μL and store at 80ºC

  1. Take the competent cells, E. coli DH5α, from the storage at 80ºC and defrost them on ice

  2. Add 1 μL of plasmid solution to the 100 μL cell tube or add 100 μL of cells to the 10 μL ligation tube

  3. Incubate on ice for 30 minutes

  4. Heat-shock the cells at 42ºC for 45 seconds

  5. Add 1 mL of SOC rich media (supplemented SOB media with 0’36% of glucose) and incubate at 37ºC for 1 hour

  6. Plate the culture on LB-agar plate overnight

  1. Grow a single Pseudomonas putida colony in LB overnight at 30ºC and 180 rpm

  2. Harvest the cells in a 1’5 mL tube by centrifugation (30 seconds at 13000 rpm) and resuspend in 1 mL of sucrose 300 mM. Do it twice

  3. Resuspend the cells in 100 μL of sucrose 300 mM

ELECTROPORATION

  1. Use 100 μL of electrocompetent cells for electroporation

  2. Add on ice 500 ng of the plasmid pTNS2 (allow the transposition of the plasmid pUC18SfiminiTn7BBGm) and 500 ng of the derivative plasmid of the pUC18SfiminiTn7BBGm

  3. Transfer the reaction mix to 2 mm electroporation cuvette that has got cold previously

  4. Electroporate at 2’5 kV, 25 μΡ and 200 Ω with a pulse of 5 ms

  5. Add 1 mL of SOC media and incubate at 30ºC for 1 hour

  6. Plate the culture on LB-agar plate overnight at 30ºC

TEST THE TRANSPOSITION

  1. Do a colony PCR like a simple PCR, but adding a colony instead of a template DNA and using the primers VR and VF2

  2. Do a electrophoresis of the colony PCR at 1'5% agarose gel

  3. Reveal the agarose gel

  1. Grow the donor strains, the acceptor strains and the strain that contains the conjugative plasmid pRK2013 in LB overnight

  2. Mix 300 μL of each strain (donor, acceptor and conjugative) in a 1’5 mL tube and centrifuge at 13000 rpm for 1 minute

  3. Wash the pellet with 1 mL of LB and centrifuge at 13000 rpm for 1 minute twice

  4. Resuspend the pellet with 100 μL of LB

  5. Spot 100 μL of the mixed cells onto a LB-agar plate and incubate for 6 hours at the adequate temperature

  6. Take some biomass of the spot and plate onto a LB-agar plate with the specific antibiotics of the donor plasmid and incubate overnight

  1. Reaction Mix

  2. PCR machine program

  1. Prepare the reaction mix

  2. Incubate at 37ºC for 2 hours

  1. Reaction Mix to get the two DNA fragments of the sequence that we want to mutagenize (one of the primers has the mutagenic sequence)

  2. PCR machine program

  3. Test the length of the PCR products in an agarose gel, excise the DNA fragment of interest and purify the agarose slice containing the DNA fragment

  4. Run 5 μL of the purified DNA fragment solution in an agarose gel to compare the concentration of DNA for each pair of fragments

  5. Equal the concentration of each pair of DNA fragments

  6. Reaction mix to get the full sequence with the specific mutation

  7. Do the same PCR machine program