Team:Groningen/Experiments

iGEM Groningen 2019 :: Attributions

Experiments

Recipes

Media
Name Components Use
LB 15.5 g/L L broth (Formedium) Growth of E. coli
LBV / LB3 LB medium supplemented with 30 g/L NaCl Growth of V. natriegens
BHI v2 37 g/L BHI broth (Difco) v2 salts Growth of V. natriegens
BHI v3 37 g/L BHI broth (Difco) v3 salts Growth of V. natriegens
LB v2 LB medium supplemented with v2 salts Growth of V. natriegens
LB v3 LB medium supplemented with v3 salts Growth of V. natriegens
V. natriegens minimal medium 2 g/L Na D-glucuronate
1.3 g/L MgSO4·7 H2O
0.75 g/L KCl
5.3 g/L (NH4)2HPO4
15 g/L NaCl
Growth of V. natriegens
M9 11.28 g/L 5 x M9 salt base (Formedium)
0.4 % glucose
2 mM MgSO4
0.1 mM Cacl2
0.3 µM thiamine·HCl
Growth of E. coli in CRISPR screen
M9 his M9 medium supplemented with 3 mM L-histidine Growth of E. coli in CRISPR screen
M9v2 M9 medium supplemented with v2 salts Growth of V. natriegens in CRISPR screen
M9v2 his M9v2 medium supplemented with 3 mM L-histidine Growth of V. natriegens in CRISPR screen
V. natriegens recovery medium BHIv2 supplemented with 680 mM sucrose Recovery of V. natriegens after transformation
Printing medium 0.5 mM Cacl2 Plates for printing with bioink

Media supplements

Name Amount Use
Agar 10 g/L Agar plates
v2 salts L204 mM NaCl
4.2 mM KCl
23.14 mM MgCl2
Growth of V. natriegens
v3 salts 475 mM NaCl
9.7 mM KCl
23.14 mM MgCl2
Growth of V. natriegens
Kanamycin E. coli: 50 µg/ml
V. natriegens: 250 µg/ml
Antibiotic selection
Chloramphenicol E. coli: 25 µg/ml
V. natriegens: 25 µg/ml
Antibiotic selection
Ampicillin E. coli: 100 µg/ml
V. natriegens: 70 µg/ml
Antibiotic selection
Tetracycline E. coli: 10 µg/ml
V. natriegens: 4 µg/ml
Antibiotic selection
Anhydrotetracycline 500, 250, 125 or 62.5 ng Induction of promoter
L-Arabinose 1, 0.5, 0.1, 0.05 or 0.01 (w/v) % Induction of promoter
Mannose 1, 0.5, 0.1 or 0.05 (w/v) % Induction of promoter

Other recipes

Name Components / Instructions Use
10 x TAE 48.4 g/L Tris base
17.4 M concentrated acetic acid
3.7 g/L EDTA
Buffer for gel electrophoresis
Agarose gel 1 % Agarose dissolved in TAE by heating
10000 x diluted DNA stain stock
Gel for gel electrophoresis
V. natriegens electroporation buffer 680 mM sucrose
7 mM KPi
Preparation of electrocompetent V. natriegens
Sorbitol 1 M sorbitol
Preparation of chemically competent V. natriegens
Calcium chloride 0.1 M Cacl2 Preparation of chemically competent E. coli
Calcium chloride, glycerol 0.1 M Cacl2
15 % glycerol
Preparation of chemically competent E. coli
Sodium Citrate 0.1 M Na Citrate Resolubilization of solidified bio-ink

Procedures

1) Molecular genetic methods

Gel electrophoresis

  1. Make an agarose gel using an adequate mold and comb
  2. Load 10 µl of the sample in the pocket alongside the DNA ladder
  3. Apply 120 V for 30 min
  4. Image the gel using gel documentation machine

Digestion ligation cloning

  1. Design the cloning procedure using the Benchling Assembly tool
  2. Digest the desired parts with the respective enzymes according to the fabricant’s instructions
  3. Inactivate the restriction enzymes
  4. Ligate all parts according to the fabricant’s instructions
  5. Inactivate the T4 ligase
  6. Transform 5 µl of the ligated construct according to protocol
  7. Select clones and confirm by cPCR and sequencing

Preparation of electrocompetent V. natriegens cells

  1. Dilute an overnight culture 1:200 in LBv2 and culture to an OD600 of 0.4 – 0.6 (use 1.5 ml culture per aliquot)
  2. Pellet cells 3 min at 8000 rpm
  3. Resuspend the cells in 1 ml of electroporation buffer
  4. Repeat steps 2 and 3
  5. Pellet cells and resuspend in electroporation buffer (30 µl per aliquot)
Electroporation of V. natriegens cells
  1. Add 300 ng of DNA to electrocompetent cells and transfer into 1 mm electroporation
  2. Electroporate at 900 V, 25 µF, 200 Ω
  3. Recover with 1 ml recovery medium and culture for 1.5 h at 37 °C
  4. Plate on LBv2 plates with according antibiotic marker
Preparation of chemically competent V. natriegens cells
  1. Grow overnight at 30 °C, 275 rpm in BHIv2
  2. Dilute the overnight culture 1:200 in BHIv2 (100 ml) and grow at 37 °C in a baffled flask to an OD600 of 0.4
  3. Pellet the cells at 6465 rpm for 10 min
  4. Resuspend in 3.25 ml of prewarmed 1 M sorbitol and incubate for 1 h
  5. Use 200 µl per aliquot
  6. Use the same day or freeze in -80 °C
Preparation of chemically competent V. natriegens cells
  1. Grow overnight at 30 °C, 275 rpm in BHIv2
  2. Dilute the overnight culture 1:200 in BHIv2 (100 ml) and grow at 37 °C in a baffled flask to an OD600 of 0.4
  3. Pellet the cells at 6465 rpm for 10 min
  4. Resuspend in 3.25 ml of prewarmed 1 M sorbitol and incubate for 1 h
  5. Use 200 µl per aliquot
  6. Use the same day or freeze in -80 °C
Chemical transformation (V. natriegens)
  1. Thaw chemically competent cells on ice
  2. Add approx. 50 ng of DNA (not more than 10 % of the cell Volume) and flick to combine
  3. Incubate 10 min on ice
  4. Transfer to 42 °C water bath for 2.5 min
  5. Recover with 2.5 ml prewarmed recovery medium and let outgrow for 2 h in 37 °C
  6. Plate on LBv2 plate with respective antibiotic
Colony PCR (cPCR)
  1. Pick a colony using a toothpick
  2. Use the toothpick to first squeeze the colony at a bottom of a PCR tube and then inoculate liquid culture
  3. Add the PCR reaction mix and start the PCR reaction according to the fabricant’s instructions
  4. Analyze the PCR products using gel electrophoresis
CRISPR
  1. Target selection and design
    The gene to knock out was selected based on literature on auxotrophic E. coli strains. gRNA binding sites were designed using the Synthego software. We selected one gene to knockout and three gRNA targets. For homologous recombination a 1.5 kb long part up- and downstream of the HisD gene were selected and brought together.
  2. CRISPR plasmid cloning
    The pJOE8999 plasmid was digested using BsaI restriction enzyme and verified on an agarose gel. pJOE8999 contains the Cas9 enzyme under the control of the mannose inducible promoter, gRNA scaffold with two BsaI restriction sites and kanamycin resistance marker. The gRNAs were ordered as single strand nucleotides from IDT and annealed by cooling 0.1 °C/s from 95 °C to 25 °C. The annealed insert was designed in such a way that it would match the sticky ends created by the BsaI enzyme. The gRNA insert was ligated into the plasmid using the ligation protocol. The correct construct was confirmed by sequencing.
  3. Homologous part
    The homologous part of 3 kbp was ordered as gBLOCKS from IDT. This double stranded DNA fragment was shortened to 1.5 kbp and 600 bp respectively using PCR and according primers. The PCR was conducted using Q5 enzyme and following the fabricant’s instructions. Successful PCR was validated with gel electrophoresis and the fragments were isolated using Qiagen’s PCR purification kit.
  4. CRISPR knockout
    The constructed plasmid and homologous part were transformed into E. coli and V. natriegens using the respective protocols and plated on either LB or LBv2 agar plates supplemented with kanamycin and the inducing agent mannose (0.1 %) to encourage Cas9 expression. Clones that have appeared within 48 h in 30 °C were plated on LB or LBv2 agar plates without the selection marker kanamycin to eliminate the plasmid. Since this plasmid contains a heat sensitive origin of replication usually the bacteria would be incubated in 50 °C. However, we decided against this because of V. natriegens’ sensitivity to heat.
  5. Screening
    Colonies growing on non-selective agar plates were replated on M9 agar plates either with or without histidine. If a successful CRISPR mediated HisD knockout leads to a histidine auxotrophic strain the modification can be identified if the clone was able to grow only on the plate that had been supplemented with histidine.
Synthetic promoter library (SPL) construction
  1. Target selection and design
    The promoters pTET (BBa_R0040) and pBAD (BBa_K808000) were selected for optimization because they have been reported to be less inducible in V. natriegens than reported in E.coli. The SPL was designed following the work of 2013 iGEM DTU-Denmark team.
  2. SPL cloning
    Both promoters were constructed to control the expression of mCherry. The SPL was achieved by PCR of these “native” constucts with randomized primers that yield an arbitrary sequence in the promoter region. The linear PCR product was circulized with the digestion ligation method. The random plasmids were transformed in both E. coli and V. natriegens.
  3. Screening
    Clones were picked and recultured. In an initial screening all clones were measured for fluorescence of mCherry after induction with anhydro tetracycline or arabinose. Clones with at least five times inducibility were selected and remeasured for mCherry fluorescence in duplicate. Selected clones were characterized in different inducer concentrations in a time course measurement.
2) Production of QR code pattern

Etching

  1. Seed a lawn of bacteria on the plate: leave 15 ml of culture with OD600 of 0.2 for 15 min. Then remove excess liquid and let dry for 10 more minutes
  2. Using a CNC laser plotter machine (400 - 460 nm, 500 mW) at a speed of 250 mm/min or 550 mm/min and density of 12 or 14 lines per mm, etch the desired image into the bacterial lawn (variables will vary depending on organism used, these values have worked for us with E. coli and V. natriegens respectively)
  3. Let the plate outgrow at 37 °C for 24 h and admire the beautiful image in etched into the bacterial lawn

Stamping

  1. 3D print a stamp (use QRoningen software)
  2. Dilute fresh culture to an OD600 of 0.15 and dip the stamp in it
  3. Let excess liquid flow back and let stamp dry for 15 min
  4. Touch the stamp to an agar plate (you can stamp around five plates after one another)
  5. Do not move the plate and let it dry for 10 more minutes
  6. Incubate the plate overnight at 37 °C and enjoy a gorgeous bacteria stamp

Bioink

  1. Pellet an overnight culture at 4000 rpm for 3.5 min
  2. Resuspend carefully in LB or LBV supplemented with alginate.

Viability in bioink

  1. Print a drop of 100 µl from the bio-ink in the center of a printing medium plate
  2. Leave the plate for 6, 24 and 72 h on the benchtop
  3. Resolubilize the drop after the given incubation time in 0.1 M sodium citrate solution, make a serial dilution and use 10 µl each to plate on a LB or LBV plate
  4. Let grow overnight and count the appeared colonies to determine CFU

Bioprinting

  1. Prepare fresh bio-ink and printing plates (supplemented with Cacl2) and the bioprinter
  2. Create the gcode for your QR code using QRoningen software
  3. Start the bioprinter to run your gcode
  4. Incubate plates at 37 °C
3) Measurements

Fluorescence in liquid medium

  1. Grow overnight cultures of the bacteria you want to measure
  2. Inoculate in enough fresh growth medium in the morning. If needed supplement with inducers at different concentrations.
  3. Transfer 200 µl / well to 96-well plate. Remember to make a duplicate measurement. Measure OD600 and the fluorescence of your reporter in a microtiter plate reader.
    Type Absorption / Emission
    mCherryex.: 580 nm, em.: 630 nm
    RFPex.: 520 nm, em.: 580 nm
    GFPex.: 485 nm, em.: 528 nm
  4. Let cultures grow in 37 °C
  5. Transfer to 96-well plate at desired timepoints to quantify the development over time (e.g. 4 and 8 h) and measure
Fluorescence in solid culture
  1. Produce a fresh plate with cultures, either in bio-ink or regular agar plates
  2. Measure fluorescence of your reporter using the Typhoon FLA 9500 biomolecular imager.
    Type Excitation wavelength
    mCherryAlexa Fluor 532 nm excitation wavelength
    RFPCy3 532 nm excitation wavelength
    GFPAlexa Fluor 478 nm excitation wavelength

Data processing (solid)

  1. Process the image using ImageJ
  2. Select area of choice that contains the fluorescent reporter
  3. Determine average intensity in samples and controls
Data processing (liquid)
  1. Process data in any data processing software (e.g. Excel)
  2. Normalize fluorescence with OD600 (Fnorm = F / OD600)
  3. Determine foldchange to your control (FC = Fnorm / Fnorm,control)
  4. Plot fluorescence against time and inducer concentration if applicable