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

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

Digestion ligation cloning

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

Preparation of electrocompetent V. natriegens cells

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)
Pellet cells 3 min at 8000 rpm
Resuspend the cells in 1 ml of electroporation buffer
Repeat steps 2 and 3
Pellet cells and resuspend in electroporation buffer (30 µl per aliquot)

Electroporation of V. natriegens cells

Add 300 ng of DNA to electrocompetent cells and transfer into 1 mm electroporation
Electroporate at 900 V, 25 µF, 200 Ω
Recover with 1 ml recovery medium and culture for 1.5 h at 37 °C
Plate on LBv2 plates with according antibiotic marker

Preparation of chemically competent V. natriegens cells

Grow overnight at 30 °C, 275 rpm in BHIv2
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
Pellet the cells at 6465 rpm for 10 min
Resuspend in 3.25 ml of prewarmed 1 M sorbitol and incubate for 1 h
Use 200 µl per aliquot
Use the same day or freeze in -80 °C

Preparation of chemically competent V. natriegens cells

Grow overnight at 30 °C, 275 rpm in BHIv2
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
Pellet the cells at 6465 rpm for 10 min
Resuspend in 3.25 ml of prewarmed 1 M sorbitol and incubate for 1 h
Use 200 µl per aliquot
Use the same day or freeze in -80 °C

Chemical transformation (V. natriegens)

Thaw chemically competent cells on ice
Add approx. 50 ng of DNA (not more than 10 % of the cell Volume) and flick to combine
Incubate 10 min on ice
Transfer to 42 °C water bath for 2.5 min
Recover with 2.5 ml prewarmed recovery medium and let outgrow for 2 h in 37 °C
Plate on LBv2 plate with respective antibiotic

Colony PCR (cPCR)

Pick a colony using a toothpick
Use the toothpick to first squeeze the colony at a bottom of a PCR tube and then inoculate liquid culture
Add the PCR reaction mix and start the PCR reaction according to the fabricant’s instructions
Analyze the PCR products using gel electrophoresis

CRISPR

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.
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.
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.
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.
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

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.
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.
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

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
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)
Let the plate outgrow at 37 °C for 24 h and admire the beautiful image in etched into the bacterial lawn

Stamping

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

Bioink

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

Viability in bioink

Print a drop of 100 µl from the bio-ink in the center of a printing medium plate
Leave the plate for 6, 24 and 72 h on the benchtop
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
Let grow overnight and count the appeared colonies to determine CFU

Bioprinting

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

3) Measurements

Fluorescence in liquid medium

Grow overnight cultures of the bacteria you want to measure
Inoculate in enough fresh growth medium in the morning. If needed supplement with inducers at different concentrations.
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

mCherry ex.: 580 nm, em.: 630 nm

RFP ex.: 520 nm, em.: 580 nm

GFP ex.: 485 nm, em.: 528 nm
Let cultures grow in 37 °C
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

Produce a fresh plate with cultures, either in bio-ink or regular agar plates
Measure fluorescence of your reporter using the Typhoon FLA 9500 biomolecular imager.

Type Excitation wavelength

mCherry Alexa Fluor 532 nm excitation wavelength

RFP Cy3 532 nm excitation wavelength

GFP Alexa Fluor 478 nm excitation wavelength

Data processing (solid)

Process the image using ImageJ
Select area of choice that contains the fluorescent reporter
Determine average intensity in samples and controls

Data processing (liquid)

Process data in any data processing software (e.g. Excel)
Normalize fluorescence with OD₆₀₀ (F_norm = F / OD₆₀₀)
Determine foldchange to your control (FC = F_norm / F_norm,control)
Plot fluorescence against time and inducer concentration if applicable

Type	Absorption / Emission
mCherry	ex.: 580 nm, em.: 630 nm
RFP	ex.: 520 nm, em.: 580 nm
GFP	ex.: 485 nm, em.: 528 nm

Type	Excitation wavelength
mCherry	Alexa Fluor 532 nm excitation wavelength
RFP	Cy3 532 nm excitation wavelength
GFP	Alexa Fluor 478 nm excitation wavelength

Team:Groningen/Experiments