Team:UPO-Sevilla/Protocols

PROTOCOLS

Plasmid DNA extraction

  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)

DNA electrophoresis

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

Ligation

  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

Generate competent cells

  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

Heat-shock transformation

  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

Generate electrocompetent cells

  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 and transposition

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

Triparental mating

  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

PCR

  1. Reaction Mix

  2. PCR machine program

Digestion

  1. Prepare the reaction mix

  2. Incubate at 37ºC for 2 hours

Site-directed mutagenesis using overlap extension PCR

  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

Dilution series-based growth curves

  1. Plate the strains of Pseudomonas putida KT2442 and incubate at 30ºC overnight

  2. Grow a single colony in 3 mL of LB without antibiotics at 30ºC and 180 rpm overnight

  3. Dilute 20 times the culture in 1 mL of LB and measure the OD600

  4. Adjust the OD600 to 0’1 for 6 mL of LB

  5. Do 9 serial dilutions with a dilution factor of 6 (take 1 mL of the culture and mix with 5 mL of LB). Induce the expression in this step, if it would be necessary

  6. Prepare a microtiter dish for each strain and condition. Load the first and the last column with 150 μL of the media and 150 μL of the dilutions in the rest of columns, serially

  7. Incubate the microtiter dish at 25ºC and 150 rpm for 20 hours

  8. Measure the OD600 of the microtiter dish (planktonic growth)

  9. Remove the planktonic bacteria and wash the plate with water (submerge the plate in a tray of water and shake out the liquid). Repeat this step three times

  10. Dry the plate and add 200 μL of 0’1% crystal violet solution into each well and stain for 15 minutes at RT

  11. Discard the crystal violet solution and wash the plate in a tray with water three times

  12. Dry the plate and add 200 μL of 96% ethanol and stain for 20 minutes and 600 rpm at RT

  13. Measure the OD620 of the microtiter dish (biofilm growth)

Beta-galactosidase assay

  1. Plate the strains of Pseudomonas putida KT2442 that contains the plasmid with lapZ and the promoter that we want to test, and incubate at 30ºC overnight

  2. Grow a single colony in 3 mL of LB with the plasmid antibiotic at 30ºC and 180 rpm overnight

  3. Dilute 300 times the culture in 3 mL of LB with the appropriate antibiotic. Induce in this step, if it would be necessary

  4. Incubate at 30ºC for 24 hours

  5. Prepare the reaction mix: 705 μL of buffer Z + β-mercaptoethanol, 30 μL of Chloroform, 15 μL of SDS (Sodium Dodecyl Sulfate) and 50 μL of culture diluted 20 times (1140 μL of phosphate buffer 1x and 60 μL of culture)

  6. Measure the OD600 of the diluted culture

  7. Mix the reaction mix by vortex and cool down at 30ºC. Add 200 μL of ONPG (ortho-Nitrophenyl-β-galactoside), mix by vortex and incubate at 30ºC

  8. Add 500 μL of NaCO3 1M, when the reaction mix becomes yellow, and write down the time

  9. Centrifuge at 10000 rpm for 15 minutes and measure the A420 of the reaction mix

Congo Red binding assay

  1. Plate strains of Pseudomonas putida KT2442 and incubate at 30ºC overnight

  2. Grow a single colony of each strain in 3 mL of LB without antibiotics at 30ºC and 180 rpm overnight

  3. Take a 10 micro liters drop of the saturated inoculum of each strain and put it on a LB agar plate supplemented with Congo Red at a final concentration of 40 mg L-1

  4. Incubate at 30ºC for 48 hours

  5. Take pictures of the plates

Swimming assay

  1. Plate strains of Pseudomonas putida KT2442 and incubate at 30ºC overnight

  2. Take a single colony of each strain with the help of a toothpick and put it on swimming plates (0,3% Bacto-agar)

  3. Incubate at 30ºC for 12 hours

  4. Take digitally pictures of the halos

c-diGMP levels evaluation

  1. Transform strain of Pseudomonas putida with the pCdrA::gfpc plasmid.

  2. Grow a single colony of each transformed bacterium in 3 mL of LB without antibiotics at 30ºC and 180 rpm overnight

  3. Dilute each culture 250-500 times in 10% LB

  4. Prepare 96-well microtiter microplates with the diluted cultures

  5. Incubate at 30ºC and 550 rpm till middle exponential phase

  6. Dilute the cultures 100 times in 150 micro liters of the same medium in the 96-well microtiter microplates

  7. Incubate for 23 hours in the same conditions in a Tecan Spark 10M fluorimeter

  8. Measure absorbance each 15 minutes at 600nm and fluorescence at an exciting wavelength of 395 nm and an emitting wavelength of 509 nm

Bacterial growth measurement

  1. Plate strains of Pseudomonas putida KT2442 and incubate at 30ºC overnight

  2. Grow a single colony of each strain in 10 ml of the medium. Do it 3 times in order to have 3 biological replicates of each physiologic assay. Incubate them at 30ºC and 180 rpm overnight

  3. Centrifuge at 4000 rpm for 5 minutes Discard supernatant and resuspend in 10 ml of NaCl-P 1X Buffer. Repeat this step three times

  4. Prepare a 96-well microtiter microplate with 148,5 μl of the media and 1,5 μl of the bacteria in each well. Use wells of the borders to do blank measurements and standardize the resuls

  5. Put the microplate inside the fluorimeter and star the program. It will measure DO600 each 15 minutes for 23 hours at 30ºC

Adhesion assay

  1. Plate strains of Pseudomonas putida KT2442 and incubate at 30ºC overnight

  2. Grow a single colony of each strain in 3 mL of LB without antibiotics at 30ºC and 180 rpm overnight

  3. When cells reach mid-exponential phase (A600= 0.2-0.3), dilute the LB cultures of the selected strains (102 – 104) in LB samples

  4. Transfer diluted cultures to a 96-well microtiter polystyrene microplate

  5. Wait 30 minutes to allow attachment of the cells

  6. Wash twice with 150 μl LB to remove planktonic cells and add 50 μl LB to the wells

  7. Incubate at room temperature for 3 hours

  8. Video record for 1 minute

Pellicle assay on crystal tubes

  1. Plate strains of Pseudomonas putida KT2442 and incubate at 30ºC overnight

  2. Grow a single colony of each strain in 5 mL Bacto-tryptone 10 g/L. 2 biological replicates of each one. Incubate at 30ºC and 180 rpm overnight

  3. Take pictures of the tubes