Team:Groningen/Protocols

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Protocols

Protocols

Cloning Quality control Measurements List of solutions
PCR Colony PCR Fermentation Media
Plasmid Isolation Restriction analysis Buoyancy test Chemicals
Restriction Metal uptake assay for E. coli
Annealing synthetic oligo's
Ligation
Making competent cells
Transformation

Cloning

PCR

21 μL mastermix*
1 μL forward primer
1 μL reverse primer
1 μL template
1 μL Taq polymerase

*master mix contains:
100 μL Taq NH4
8 μL dNTP's
80 μL MgCl
652 μL MilliQ water

PCR Reaction

  • Hotstart
    • 95 °C, 2 min.
  • 25 cycles
    • 95 °C, 30 sec.
    • 61 °C, 20 sec.
    • 72 °C, 1.5 min.
  • End
    • 72 °C, 10 min.
    • 4 °C, ∞

Plasmid isolation

(NucleoSpin® Plasmid, Machery nagel])
(GeneElute™ Plasmid Miniprep Kit, Sigma-Aldrich)

Restriction

  • Mix
    • 1 μL 10x fast digest buffer (Fermentas)
    • 0.5 μL Enzyme A*
    • 0.5 μL Enzyme B*
    • 8 μL DNA to be digested**
  • Incubate 0.5 - 1 h @ 37 °C (Fast digest do not require long incubations, when using conventional enzymes 1 h. should be maintained)
  • Purify cut plasmid using PCR clean up kit

* a combination of two of the following (when using biobrick standard) SpeI, EcoRI, PstI and/or XbaI
** When digesting vectors, bring the digested vectors to 1% agarose gel and cut out with scalpel, purify using gel purification kit (NucleoSpin® Extract II, Machery nagel or Zymoclean™ Gel DNA Recovery Kit) to an end volume indicated by the kit (End volume determines concentration, variations are possible) (alternatively, Phosphatase treatment of linearized vector)

Annealing synthetic oligo’s

Protocol #2

Phosphorylation

  • Mix
    • 1 μL PNK
    • 1 μL T4 DNA Ligase buffer
    • 8 μL Oligo’s (FW and REV seperate reactions)

100x diluted oligo’s (1 μM)

  • Procedure
    • 30 min 37 °C
    • 20 min 65 °C

Annealing

  • Mix
    • 8 μL FW Phosphorylation mix
    • 8 μL REV Phosphorylation mix
    • 4 μL 10 mM NaCl low?
    • 20 μL MilliQ
  • Procedure
    • Put reactions in (close to) boiling water
    • Let it cool down naturally to ~35 °C

Restriction vector

  • Mix
    • 1 μL EcoRI
    • 1 μL XbaI
    • 2 μL Fast digest buffer (10x)
    • 8 μL Vector DNA
    • 8 μL MilliQ
  • Procedure
    • 37 °C, 30 min. incubation
    • Heat inactivation EcoRI and XbaI

5 min. 80 °C 15 min. 65 °C

Ligation of Metal sensitive promoters in vector

  • Mix
    • 2 μL T4 Ligase Buffer
    • 0.5 μL T4 Ligase
    • 1 μL Restriction product (unpurified)
    • 1 μL Annealed oligo’s
    • 18 μL MilliQ
  • Procedure
    • Incubation 16 °C, 1 hour
    • Heat inactivation Ligase

65 °C, 10 min.

Protocol #3

Restriction vectors

  • Mix
    • 2 μL Tango digest buffer (Fermentas)
    • 16 μL vector
    • 1 μL EcoRI (Non-fast digest)
    • 1 μL SpeI (Non-fast digest)
  • Incubate 1.5 h @ 37 °C
  • Put to 1% agarose (1xTBE)
    • Isolate ~3000 bp vector
    • Check for presence of cut out ccdB death gene (~600 bp)
  • Purify vector using gel purification kit
    • End volume 30 μL H2O (MilliQ)
    • Determine concentration using nanodrop
    • Store to 4 °C until ligation

Phosphorylation of 5' ends & hybridization [1]

  • (SENSE) Mix:


  • (ANTI-SENSE) Mix:
  • Incubate @ 37 °C for 1.5 hours.
  • Mix
    • 10 μL Sense mixture
    • 10 μL Anti-sense mixture
    • 3 μL 0.5 M NaCl
  • Place in boiling water for 3 min., and allow the reaction to cool to room temperature.

Ligation

  • Mix
    • 1 μL T4 ligase buffer
    • 7.5 μL restricted vector (purified from gel)
    • 1 μL annealing mix
    • 0.5 μL T4 ligase
  • Incubate
  • 1h RT
or
  • ON @ 4 °C

Transformation

  • Add 5 μL of ligation mixture to 50 μL of TOP10 chemically competent cells
  • Heatshock, 45 sec. 42 °C
    • + control: 1 μL pSB3K3-high or pSB1AC3-high plasmid, - control: 1 μL MilliQ
    • Alternatively a single cut plasmid can be taken as a ligation control
    • Alternative - control: 1 μL pSB1AC3 or pSB3K3 carrying ccdB deathgene
  • Incubate 1 h @ 37 °C, 200 RPM
  • Plate out on LB-agar + Kanamycin (30 μg/ml for pSB3K3) or Ampicillin (100 μg/mL for pSB1AC3)
    • Plate out 50 μL & 200 μL of cell suspension
  • Grow ON @ 37 °C

Checking transformations

  • See if - control is empty for functioning antibiotics and death gene
  • See how many colonies on + control for functioning competent cells
  • See how many selfclosers and compare to samples (>10x on sample vs. selfcloser)
  • If enough transformants, inoculate 3 - 5 colonies in an ON culture
    • Alternatively perform colony PCR


Ligation

  • Ligation
    • μL ligation buffer
    • 1 μL ligase
    • μL vector
    • μL insert
    • Incubate

Making competent cells

Competent cells: TOP10 & DB3.1

  • 10 mL ON culture is used to inoculate LB, 100 μL ON culture per 20 mL*
  • Cultures are grown @ 37 °C until an OD600 of 0.2 ~ 0.3 is reached.
  • Cultures are spinned down 5 min. @ 4000 rpm, 4 °C
  • Supernatant is removed and pellet (per 20 mL culture) is resuspended in 5 mL chilled 0.1 M CaCl2
    • Suspension is incubated on ice for 10 min.
  • Suspensions are spinned down 5 min. @ 4000 rpm, 4 °C
  1. Supernatant is removed and pellet is resuspended in 1770 μL chilled 0.1 M CaCl2 and supplemented with 230 μL 87% glycerol prior to making aliquots.
  2. Cells are divided in 50 μL aliquots
  3. Cells are snapfrozen in liquid nitrogen and stored @ -80 °C

* Cultures should be grown in the ratio 1:5 (medium:air), so 10 mL culture in a 50 mL greiner tube.

Transformation

  • Add 10 uL of ligation mixture or 1 uL isolated plasmid to competent cell aliquot
  • Incubate on ice for 15 - 30 min.
  • Heatshock 45 sec. @ 42 °C or 5 min. 37 °C
  • Let cells relax on ice for 1 - 2 min.
  • Add LB 200 μL (WJP) or 800 μL
  • Incubate 37 °C, 250 RPM for 1 h.
  • Plate out, or spinned cells down and resuspend prior to plating out

Quality control

Colony PCR

  • Put colony in 1 μL MilliQ water
  • Put colony suspension in microwave for 1 min. 1000 W
  • Use this as DNA template
  • TODO

Restriction analysis

Measurements

Fermentation

Performed in 2L autoclavable fermenter with dished bottom vessel stirred fermenter

  • Autoclave closed fermenter system
  • Inoculate 1.3 L LB (+100 µL Y30 antifoam) with 20 mL ON culture was used to
    • Airflow rate of 1 vvm
    • pH @ 7 (by addition of 4 M NaOH or 1 M HCl)
    • Temperature @ 37 °C
    • Agitation 400 to 800 RPM*
    • Oxygen concentration >50%
  • Take samples every 0.5 to 1 h. to determine optical density at 600 nm
  • 50 mL samples in every growth phase (pre-exponential, early exponential, exponential, late exponential, steady state)
  • Spin samples down 35 min., 1000 RPM and remove supernatant
  • Continue Buoyancy test

*6-bladed flat disc turbine (Rushton type) impeller (60 mm diameter) at the bottom to disperse the bubbles coming from the sparger underneath and a 3-bladed marine impeller, vortex (60 mm diameter) halfway the broth volume to create an axial flow.

Buoyancy test

Continued from cultures (flask or fermenter) after centrifugation

  • Resuspend pellet in 1 - 5 mL saline solution
  • Determine OD600
  • Dilute suspension to OD600 1.5 with saline solution
  • Put homogeneous suspension in tubes, take care of descent lighting from behind (day light is best)
  • Record decrease of buoyancy (matter of hours in fermentation cultures, days in shakeflask cultures)

Metal uptake assay for E. coli (according to Kostal et al. 2004)

  • Grow o/n culture of E. coli WT @ 30dg
    • Use E. coli + control vector, E. coli + pArsR-RFP, E. coli + pLac-fMT
  • Inoculate day culture 1:50, grow in 1L TB-Amp (100ml per time/[As(III)] sample)
    • Take OD600 samples every 1-1.5 hrs of E. coli + pLac-fMT.
    • Induce E. coli + pLac-fMT at OD600 ~0.6 with 0.5mM IPTG.
  • Harvest the cells in stationary phase (after ~30hr) by spinning down @ 4000rpm for 20min in Sorval centrifuge.
  • Wash 2 times with TB74S buffer.
  • Resuspend in prewarmed (30 C) TB74S buffer upto a OD of ~25
    • Take a 1ml sample in small alluminium boxes and dry @ 104dg for >4 hrs.
    • Afterwards measure the dry weight of the sample and calculate the weight/volume of the entire sample.
  • For the concentration range:
    • Incubate 5 samples (of same time point) for 1hr @ 30dg with 0μM, 10 μM, 20 μM, 50 μM and 100 μM As(III).
  • For the concentration range:
    • Incubate 5 samples (of same concentration) @ 30dg with 10 or 100μM As(III) for 0, 10, 20, 40, 60 min.
  • Harvest cells by spinning down.
  • Wash the cells with TB74S buffer.
  • Resuspend in 10ml ddH2O.
  • Dry sample @ 65dg for 2days.
  • Store at fridge or -80dg freezer.
  • Determine the amount of As(III) in the cell at different stages and at different uptake concentrations using ICP-MS.

Analysis of arsenic concentration of ICP-MS

  • Weigh 0.1g dried E. coli cells.
  • Add 5 ml 65% nitric acid.
  • For destruction the following microwave program was used:
Stage 1 Stage 2
Power(max) 1200 1200
Power(%) 100 100
Ramp(min) 15 15
Hold(min) 0 30
Temp(graden C) 140 210


  • Let the samples cool down.
  • Dilute the samples by adding ddH2O upto 50 ml.
  • If needed, spin down 15min @ 4000rpm in a Sorvall centrifuge.
  • Measure the arsenic concentration by ICP-MS using both the standard mode (shows interference peak from multi-atomic molecule argon-chloride with the arsenic peak) and the collusion cell technology mode (doesn’t show the interference peak but has a 10x lower resolution than standard mode).
    • Use a standard curve between 0-10µg As/L and 0-100µg As/L using a certified 1000ppm (mg/L) stock

List of solutions

Media

LB(Agar)

  • 10 g (Bacto)Trypton
  • 10 g NaCl
  • 5 g Yeast extract
  • Dissolve in 1 L demi water
  • (1.5% Agar)
  • Autoclave
  • Store @ 60 °C

TB medium

  • 12g Bacto-Tryptone
  • 24g Bacto-Yeast Extract
  • 4ml Glycerol [87%]
  • dissolve in 900ml demi water
  • Separetely prepare 100 mL Kpi
    • 0.17M KH2PO4 (mw=136.09g/mol) (6.94g/300ml)
    • 0.72M K2HPO4 (mw=174.18g/mol) (7.62g/300ml)
    • dissolve in demi water
  • Autoclave and mix

Antibiotics

Ampicillin

100 mg/ml Ampicillin (1000x) Stock

  • 1 g of Ampicillin sodium salt in 10 mL of demiwater (or 50% EtOH)
  • Add NaOH or KOH to allow the Ampicillin to dissolve
  • Filter sterilize 0.2 μm filter and aliquot
  • Store -20 °C

Chloramphenicol

35 mg/ml Chloramphenicol (1000x) Stock

  • 0.35 g in 10 mL 100% EtOH
  • Filter sterilize 0.2 μm filter and aliquot
  • Store -20 °C

Kanamycin

50 mg/ml Kanamycin(1000x) Stock

  • g in 10 mL
  • Filter sterilize 0.2 μm filter and aliquot
  • Store -20 °C

Chemicals

0.1 M CaCl2

  • 0.3319 g CaCl2
  • Dissolve in 30 mL demi water

0.15 M NaCL (Saline solution, 0.9% NaCl)

  • 9 g NaCl
  • Dissolve in 1 L demi water

10x TBE buffer

  • 108 g Tris
  • 55 g Boric acid
  • 8.3 g EDTA
  • Dissolve in 1 L demi water
  • Adjust pH to 8.3

4 M NaOH

  • 160 g NaOH
  • Dissolve in 1 L demi water

~1 M HCl

  • 500 mL demi water
  • 500 mL HCl (37%, 11 M)

TB74S Buffer

  • 0.605 g Tris (5mM)
  • 8.76 g NaCl (150mM)
  • Dissolve in 1 L Demi water
  • Set pH with HCl to 7.4

Sodium Arsenite (III)

  • 100mM Na-As solution
  • filter sterilize