Team:Lund/Experiments

iGEM Lund 2019

Protocols and Experiments

Protocols

PCR (Amplification of gBlocks)

Mastermix:

 For each 20 µL reaction mixture, mix the following:

  • 2 µL dNTP mix
  • 2 µL Forward primer
  • 2 µL Reverse primer
  • 0.1 µL Phusion High Fidelity DNA Polymerase
  • 2 µL Phusion buffer
  • 10 µL MilliQ water

Thermal Cycler Settings:

  1. 98°C for 180 seconds
  2. 98°C for 20 seconds
  3. Set a gradient for 55.9°C, 57.1°C and 62.1°C for 60 seconds
  4. 72°C for 90 seconds
  5. Repeat step 1-4 for 25 cycles
  6. 72°C for 10 minutes
  7. Store at 4C° until loading

Gel Electrophoresis

Gel preparation:

  1. Weigh 1.5 gram agarose.
  2. Mix in 100 mL TAE buffer.
  3. Warm the mixture in microwave and heat until the solution is clear.
  4. Add 12 µL GelRed after the solution has cooled down.
  5. Fix comb and add the solution into the casket for the gel to solidify.

Ladder & samples:

  1. Mix 2 µL gene ruler with 2 µL loading dye and 1 µL MilliQ water for preparation of ladder.
  2. Mix 10 µL of PCR samples with 2 µL loading dye.

Electrophoresis conditions:

  1. Remove the comb and move the solidified gel to the electrophoresis set up.
  2. Fill the chamber with TAE buffer until all the wells are covered completely.
  3. Load gel and run it for 90 minutes at 100 V.

Colony PCR

Template preparation

  1. Pick colonies with inoculation loop and re-suspend in 50 μL of MilliQ water.

PCR reaction mixture

For each 20 µL reaction mixture, mix the following:

  • 2 µL dNTP mix
  • 2 µL Forward primer
  • 2 µL Reverse primer
  • 0.1 µL Dream Taq DNA Polymerase
  • 2 µL Dream Taq buffer
  • 2 µL template prepared in previous step
  • 10 µL MilliQ water

Thermal Cycler Protocol

  1. 95°C for 5 minutes
  2. 95°C for 30 seconds
  3. 59°C for 30 seconds
  4. 72°C for 45 seconds
  5. Repeat above steps for 30 cycles
  6. 72°C for 5 minutes
  7. Store at 4°C until loading

Transformations

 For TG1 and BL21-DE3 cells (Heat Shock):

  1. Add 2 μL of plasmid DNA to 50 μL cells on ice (no more than 5 μL, i.e. no more than 10% volume of cells).
  2. Swirl gently to mix the contents.
  3. Incubate on ice for 30 minutes.
  4. Transfer tubes to shaking incubator at 42oC for 90 seconds.
  5. Place samples back on ice and let them cool for 1-2 minutes.
  6. Add 800 μL of LB media.
  7. Incubate for 60 min in the shaking incubator at 37°C, for the cells to revive.
  8. Plate 75-100  μL of the culture on LB agar and incubate overnight at 37 °C.

 For E. coli Nissle cells (Electroporation):

  1. Keep 2 mm electroporation cuvettes on ice to cool.
  2. Let the electrocompetent EcN cells (-80°C) thaw completely on ice.
  3. Add 1 μL of pure plasmid DNA to 25 μL of electrocompetent EcN cells.
  4. Pipet the mixture into chilled cuvette and flick to deposit cells at the bottom.
  5. Electroporate at 1800 V for 5 ms.
  6. Add 975 μL of LB medium to the cuvette, and pipette up and down to resuspend the cells.
  7. Transfer to a culture tube and place in shaking incubator for 1 hour at 37 °C.
  8. Plate 75-100  μL of the culture on LB agar and incubate overnight at 37 °C.

Medium Preparation

LB Media (for 1 L):

  • 5 g Yeast Extract 
  • 10 g Tryptone
  • 10 g NaCl
  • Weigh ingredients individually and put them in a schott bottle with a magnetic stirrer
  • Fill with 1 L of distilled water
  • Stir until all powder is diluted

TB Media (for 1 L):

  • 12 g Peptone
  • 24 g Yeast Extract
  • 4 mL Glycerol
  • 2.3 g KH2PO4
  • 12.5 g K2HPO4
  • Weigh and later autoclave Potassium salts, glycerol separately and peptone along with yeast extract to avoid precipitation
  • Dissolve all the components in a sufficient amount of distilled water but not exceeding the total 1 L volume

Enriched Complex Media (for 1 L):

  • 20 g Peptone
  • 20 g Yeast Extract
  • 30 g Glucose
  • 13 g KH2PO4
  • 10 g K2HPO4
  • 5 g NaCl
  • 2 g (NH4)2SO4
  • 1 g MgSO4 * 7 H2O
  • 0.2 g NH4Cl
  • Weigh and later autoclave Potassium salts,  Sulfate salts, Chloride salts, glucose separately and peptone along with yeast extract to avoid precipitation
  • Dissolve all the components in a sufficient amount of distilled water but not exceeding the total 1 L volume

LB Agar ( for 1 L  approximately 45- 50 Petri Dishes):

  • 5 g Yeast Extract 
  • 10 g Tryptone
  • 10 g NaCl
  • 150 g agarose
  • Weigh ingredients individually and put them in a schott bottle with a magnetic stirrer
  • Fill with 1 L of distilled water
  • After autoclaving, cool the media and add 100 μg/ml of antibiotic of choice (ampicillin, carbenicillin, chloramphenicol) depending on the application
  • Pour into sterile Petri dishes

SDS-PAGE

Reagents:

  1. Make 50 mM sodium acetate buffer at pH 5.0 
  2. Pre-casted gel (Bio-Rad) 
  3. Loading buffer (sample buffer):
    1. 3.3 ml of TRIS 0.5 M pH 6.8 
    2. 8 ml of SDS 10% w/v 25 
    3. 4 ml of glycerol 
    4. 1 ml of bromophenol blue 0.2% w/v 
    5. 2.62 g of DTT (dithiotreithol) 
    6. Adjust volume to 20 ml using MQ-water 
  4. Electrophoresis (Running) buffer:
    1. 7.5 g of Tris 
    2. 36 g of glycine 
    3. 2.5 g of SDS 
    4. Adjust pH to 8.3 using HCl and make the final volume up to 500 ml using MQ-water. Dilute the buffer 5 times
  5. Molecular weight marker
  6. Staining solution:
    1. 1 g of Coomassie 0.2% 
    2. 200 ml of MeOH 40% 
    3. 50 ml of HAc 10% 
    4. 260 ml of MQ-water 
  7. De-staining solution:
    1. 40% methanol
    2. 10% acetic acid
    3. 50% water

Sample Preparation:

  1. Check OD and normalize all the samples to OD 2. 
  2. Resuspend pellet in 20 µl of 2x SDS loading dye. 
  3. Boil samples at 95°C for 10 min. 
  4. Load the prepared samples into the gel (Bio Rad plates, 15 well of 15μL, 4 - 20% gradient gel).
  5. After loading, fill the apparatus with the buffer solution between the glass plate until it reaches the maximum volume.

Running the Gel:

  1. Run the gel at 80 V until the dye front migrates into the running gel (~10 min).
  2. Increase to 160 V until the dye front reaches the bottom of the gel (~30 min). 

Staining & Destaining the Gel:

  1. Remove the gel from the apparatus. 
  2. Remove the spacers and glass plates, by using the metal spatula. 
  3. Place the gel in a plastic container. 
  4.  Add ~20 ml staining solution ( 50% methanol, 10% HAc & 0.1% Coomassie Brilliant Blue) and stain for 1 hour in a shaking plate. 
  5. Pour off the staining solution back to the Schott bottle.
  6.  Add ~5 mL destaining solution ( 50% Methanol & 10% HAc) and destain briefly  for ~1 min.
  7.  Pour off  the destaining solution in the active carbon containing Schott bottle. 
  8. Add ~ 10 mL of destaining solution. 
  9. Destain in a shaking plate until the gel is visibly destained (overnight). 
  10. Pour off  the destain solution in the active carbon containing Schott bottle. 
  11. Rinse with DDI H2O. 

Plamid Isolation

 The protocol according to Nucleospin plasmid kit was followed for Isolation of plasmid with low copy number.

DNA Purification

The protocol according to Nucleospin Gel and PCR clean-up kit was followed for DNA purification.

Restriction

  1. Premix the following in an eppendorf:
    • MilliQ water-15 µL
    • 10 X Buffer Tango- 2 µL
    • Plasmid to be digested- 1 µL
  2. Mix 2 µL of PscI enzyme to the mixture and keep the eppendorf on ice at all times.
  3. Mix gently and spin down for a few seconds.
  4. Incubate at 37°C for 2 hours.
  5. Inactivate PscI by incubating at 65 °C for 20 min.
  6. Cool the mixture by keeping on ice.
  7. Add 2.5 µL of 10X buffer Tango to the same eppendorf.
  8. Add 2.5 µL of enzyme NdeI and vortex briefly.
  9. Incubate at 37 °C for 2 hours.
  10. Inactivate NdeI by incubating at 65 °C for 20 min.

Ligation

  1. Mix digested gBlock and plasmid in ratio of 1:1, i.e., 50 ng of gBlock and plasmid, or in the ratio of 1:3, i.e., 60 ng gBlock and 20 ng plasmid.
  2. Add 2 µL of T4 DNA ligase buffer.
  3. Add 1 µL of T4 DNA ligase. 
  4. Add MilliQ water to make the volume upto 20 µL.

Toxic Metals stock solution

Regarding toxic metals Arsenic Standard for ICM-MS and Lead Standard for AAS were purchased from Sigma- Aldrich.

For arsenic, in 250 mL culture:

  • For 200μΜ add 7.5 ml
  • For 50μΜ add 0.99 ml
  • For 10μΜ add 0.188 ml

For lead, in 250 mL culture:

  • For 200μΜ add 10.36 ml
  • For 50μΜ add 2.59 ml
  • For 10μΜ add 0.52 ml

Sample pretreatment for testing (Eurofins ICP-MS)

  1. Centrifuge 15 μL of sample at 8000 rpm for 8 minutes.
  2. Transfer supernatant to new tubes. 

pH regulation via buffer

  1. Prepare 2 M Na2HPO4 and 1 M citric acid buffer.
  2. To obtain pH 3, add 3.42 mL of 2 M Na2HPO4 and 13.25 mL of 1 M citric acid to 250 mL of media.
  3. To obtain pH 5, add 8.22 mL of 2 M Na2HPO4 and 8.45 mL of 1 M citric acid to 250 mL of media.

Bradford

Materials:

  • Bovine Serum Albumin (BSA) (Sigma-Aldrich)
  • Coomassie Brilliant Blue G-250 (Sigma-Aldrich, catalog number: 27815 )
  • Methanol
  • Phosphoric acid (H3PO4)
  • Bradford reagent:
    1. Dissolve 50 mg of Coomassie Brilliant Blue G-250 in 50 ml of methanol and add 100 ml 85% (w/v) phosphoric acid (H3PO4).
    2. Add the acid solution mixture slowly into 850 ml of H2O and let the dye dissolve completely.
    3. Filter to remove the precipitates just before use.
    4. Store in a dark bottle at 4 °C.

Prepare Calibration curve:

  1. Prepare a 5-point calibration curve with bovine serum albumin (BSA) as standard.
  2. Make solutions of 0.2, 0.4, 0.6, 0.8 and 1.0 mg/ml BSA.
  3. Rest samples at room temperature and wait exactly 5 minutes.
  4. Measure the absorbance spectrophotometrically at 595 nm.

Sample handling:

  1. Mix 25 µl sample (BSA standard solution or your sample) with 1.0 ml of Bradford reagent.
  2. Rest samples at room temperature and wait exactly 5 minutes and then measure the absorbance spectrophotometrically at 595 nm, absorbance will increase over time.

Sample preparation for sequencing:

  1. Add 5μL of DNA 100 ng/μL.
  2. Add 5μL of each primer concentration 5 μM.
  3. Mix by inverting 6-8 times in an Eppendorf. 
  4. Forward and reverse primer should be in different tubes along with 5 μL of DNA in each.

Experiments

The first parts of experiments were the cloning and isolation of plasmids. The gBlocks with the appropriate biobricks were purchased from IDT and Eurofins. They were amplified using PCR. The PCR product was then inserted into a pUC19 vector using plasmid digestion and ligation. The plasmids were transformed into the cloning strain of E.coli TG1.  Some plasmids were purchased as a complete construct with no cloning necessary. Plasmids were isolated and transformed into E.coli BL21-DE3 cells and E.coli Nissle cells. The latter was isolated from the Mutaflor® probiotic supplement.


The presence of the proper E.coli strain out of the Mutaflor® supplement was verified with cPCR, for this experiment specific primers were designed.


For comparison of strains, all experiments were performed using both E.coli BL21-DE3 and E.coli Nissle. The transformed cells were cultivated under differing growth parameters. The parameters that were varied were induction time and pH. Cultivations were performed using shake-flasks in a shake-incubator at 37 °C. The analysis methods used to evaluate the cultivations were spectrophotometric measurements of optical cell density at 600nm, pH-measurements, SDS-PAGE, and Bradford analysis. 


The genetically engineered cells were tested for toxic metal uptake by cultivating the cells in media containing toxic metals. The concentration of metal in the media was measured at start concentration and continuously during the cultivation to detects changes in concentration. A lower metal concentration in the media would mean uptake of metals into the cells. 


Before start testing the original construct with EcN, preliminary testing of the gene construct took place using E.coli BL21(DE3) with T7 promoter.


Plasmid purification

Five gblocks were purchased from IDT, the gene for ArsR using promoter T7 and Tac, fusion protein of ArsR+ MBL using promoter T7 and genes for PbrD and pbrT using promoters T7 and Tac. Whole plasmids with the genes for PbrD+ pbrT, ArsR and MBL with the T7 promoter was purchased from IDT. An additional purchase from eurofins was made of a whole plasmid with the gene for ArsR using the Tac promoter. The whole plasmids were all delivered in a company specific vector with an ampicillin antibiotic marker. PCR was used to amplify the gblocks, using amplification primers of our own design. The PCR protocol used is described under “Protocols”. The PCR product was run through agarose electrophoresis and purified using a plasmid purification kit found under “protocols”. The purified gblocks were digested according to the digestion protocol found under “protocols”, using the restriction enzymes PscI and NdeI. The vector pUC19 was also digested using the same enzymes. The gblocks and vector was then ligated using T4 ligase while following the ligation protocol under “protocols”. The plasmids were transformed into E.coli BL21-DE3 chemically competent cells using the heat-shock protocol under “protocols”. Plasmids were transformed into E.coli Nissle electrocompetent cells following the protocol for the Nissle electroporation under “protocols”. 

Cultivation

The genetically modified cells were cultivated in LB media in shake-flasks in a shaking-incubator set at 175 rpm and 37 °C. The growth of the cells was analysed and compared to control strain of E.coli BL21-DE3 and E.coli Nissle without the inserted plasmid. The growth was measured using a spectrophotometer to measure the optical cell density at 600nm. Growth curves can be found on the results page. Two different induction times were tested 0.8 or 2 OD600nm. For the induction Isopropyl β-d-1-thiogalactopyranoside (IPTG) was used.  After induction samples were taken every hour for the monitoring of optical density and prepared for SDS-PAGE. After three constant OD readings the cultivation was terminated, since the cultures had reached stationary phase and the possibility of protein expression was minimized.

Testing protein expression

Two methods were used to analyse the protein expression; SDS-PAGE and Bradford assay. To analyze the amount of the protein of interest the SDS-PAGE samples were prepared and loaded in the set up as is described in “protocol”. To analyze the total amount of protein a bradford analysis was performed as described above in the “protocol”. 

Testing of toxic metal uptake to modified cells

Metal uptake in the genetically modified cells were tested, both using strain E.coli BL21-DE3 and E.coli Nissle. The cells were cultivated in an enriched complex media, recipe found under protocols. After 12 hours of cell growth, the cells were harvested and put into new media containing different concentrations of the metal. The cells modified to take up arsenic were added to arsenic containing media, and same for the lead uptaking cells.