From 2009.igem.org

Revision as of 22:20, 20 October 2009

Protocols

Cloning

[http://openwetware.org/wiki/PCR PCR]

Plasmid isolation

Usually performed using Miniprep kits like NucleoSpin^® Plasmid, (Machery nagel) or GeneElute™ Plasmid Miniprep Kit (Sigma-Aldrich). This is a consensus protocol.

• Spin down ON Culture in table top centrifuge, 1 min. 13.000 RPM
• Resuspend pellet, RNAse is added to degrade RNA (200 μL)
• Add lysis buffer (200 μL), to lyse the cells and to release their contents
• Add neutralization buffer (350 μL), proteins will denaturate
• Centrifuge in table top centrifuge 10 min. 13.000 RPM
• Add clear lysate in column provided in kit
• Spin down 1 min. 13.000 RPM
• Add wash buffer (usually needs EtOH to be added!) (500 - 750 μL)
• Remove flow-through and spin again to remove residual wash buffer
• Put column in clean 1.5 mL cup and add 15 - 50 μL MilliQ water or Tris buffer (pH=8.0)^*
• Incubate for 1 - 2 min.
• Spin down 1 min. 13.000 RPM

^*Less volume gives higher concentrations, supplied Tris buffer is claimed to give higher yields

Restriction

• Mix
  • 1 μL 10x fast digest buffer ([http://www.fermentas.com/ Fermentas]) or correct [http://fermentas.com/techinfo/re/5bufferplussystem.htm#Buffers conventional buffer]
  • 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) [http://www.fermentas.com/catalog/re/fastbcui.htm SpeI], [http://www.fermentas.com/catalog/re/fastecori.htm EcoRI], [http://www.fermentas.com/catalog/re/fastpsti.htm PstI] and/or [http://www.fermentas.com/catalog/re/fastxbai.htm XbaI]
^** When digesting vectors, bring the digested vectors to 1% agarose gel and cut out with scalpel, purify using gel purification kit ([http://www.mn-net.com/Products/NucleicAcidPurification/DNAcleanup/NucleoSpinExtractII/tabid/1452/language/en-US/Default.aspx NucleoSpin^® Extract II, Machery nagel], [http://www.zymoresearch.com/content/zymoclean-gel-dna-recovery-kit-d4001-d4002-d4007-d4007-d4001s Zymoclean™ Gel DNA Recovery Kit] or similar) to an end volume indicated by the kit (End volume determines concentration, variations are possible) (alternatively, [http://openwetware.org/wiki/Phosphatase_treatment_of_linearized_vector Phosphatase treatment of linearized vector])

Annealing synthetic oligo’s

Phosphorylation of 5' ends & hybridization^{[http://openwetware.org/wiki/Silver:_Oligonucleotide_Inserts[1]]}

• Mix:
  • 3 μL 100 µM (anti-)sense oligo
  • 1 μL 10 x PNK (polynucleotide kinase) buffer ([http://www.fermentas.com/catalog/modifyingenzymes/t4polynucleotidekinase.htm Fermentas Buffer A])^**
  • 2 μL 10mM ATP ^**
  • 1 μL [http://www.fermentas.com/catalog/modifyingenzymes/t4polynucleotidekinase.htm T4 polynucleotide kinase (PNK)]
  • 3 μL MilliQ
    • (for selfcloser control, do not add oligo's. Instead 6 μL MilliQ in total)
• 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.
  • Upon reaching room temperature add restricted vector (see for ratio Ligation
  • If kept at low temperature before ligation heat up the annealing mixture up to 65 °C for 1 min. to prevent the formation of multimers

^**Alternatively T4 DNA Ligase buffer can be used, already containing ATP

[http://openwetware.org/wiki/DNA_ligation Ligation]

• Mix^*
  • 1 μL [http://www.fermentas.com/catalog/modifyingenzymes/t4dnaligase.htm T4 ligase buffer]
  • 7.5 μL vector (purified from gel)
  • 1 μL Insert
  • 0.5 μL [http://www.fermentas.com/catalog/modifyingenzymes/t4dnaligase.htm T4 ligase]
• Incubate

• 1h RT

or

• ON @ 4 °C

^*This is a consensus, calculations should be performed to have the ligations be done in a 5:1 - 10:1 (Insert:Vector) mass ratio.

Making competent cells

Competent cells: [http://openwetware.org/wiki/TOP10_chemically_competent_cells TOP10] & [http://openwetware.org/wiki/E._coli_genotypes#DB3.1 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
  • + 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 [http://partsregistry.org/Part:pSB1AC3 pSB1AC3] or [http://partsregistry.org/Part:pSB3K3 pSB3K3] carrying ccdB deathgene

• 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 on LB-agar + Kanamycin (30 μg/ml for [http://partsregistry.org/Part:pSB3K3 pSB3K3]) or Ampicillin (100 μg/mL for [http://partsregistry.org/Part:pSB1AC3 pSB1AC3])
  • Plate out 50 μL & 200 μL (or 100 μL after spinning down and resuspending cells) 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

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
• PCR reaction

• Put PCR product on agarose gel

Restriction analysis

Protein expression

Growth and Purification of membrane protein obtained from E. coli (DH5α)

Culture Volume 1 L Homogenization French press Detergent DDM (Bis(4-chlorophenyl)methane) Purification Ni-NTA, FPLC

1 – Preculture.

Prepare 25 ml of bacteria culture on the day prior to the main experiment. Use LB medium with 50μg/ml amp. (1:1000 of the stock solution). Incubate over night in 37°C with shaking.

2 – Main Culture.

Use 20 ml of preculture to start main culture in 1L LB medium containing 50μg/ml amp. Incubate in 37°C with shaking till OD660=0.6, (approximately 2.5h of incubation, check the OD every hour) Add arabinose to final concentration 0.1% (10 ml of stock solution). Incubate 1 hour in 37°C with shaking. 3 – Culture Wash.

Cool off the culture on ice. Spin down the culture at 8000 rpm for 10 min in 4°C in GSA rotor.(divide 1 L volume into 3 times 400ml GSA bottles – close tightly) Wash each pellet with 40 ml ice-cold 50 mM KPi pH 7.0. Spin down the culture at 8000 rpm for 10 min in 4°C in GSA rotor.(fit into 1 GSA bottle – close tightly) Resuspend pellet in up to 12 ml 50 mM KPi pH 7.0 (no more then 13 – French press capacity) 4 – Homogenization.

Cool down all the parts of French press machine on ice and assemble it. Set the pressure on 14 MPa. Repeat the homogenization 2-3 times. 5 – ISO purification.

Spin down at 8000 rpm for 10 min in 4°C in SS-34 rotor. Collect the supernatant into 4 TLA 100.4 tubes.(not more then 3 ml into each TLA 100.4 tube) ISO’s can be stored in -80°C at this stage. Spin down at 90 000 rpm for 25 min in 4°C in ultracentrifuge (TLA 100.4 rotor). Resuspend pellet in 1 ml of 50 mM KPi pH 7.0 + 1M NaCl in 2 TLA 100.4 tube. Spin down at 80 000 rpm for 25 min in 4°C in ultracentrifuge (TLA 100.4 rotor).

6 – Solubilization.

Step A

Resuspend in 950 μl of solubilization buffer (50 mM KPi pH 8.0 + 400 mM NaCl + 20% glycerol) and 50 μl of 10% DDM in 1 TLA 100.4 tube Incubate in 4°C with shaking for 30min. Spin down at 80 000 rpm for 25 min in 4°C in ultracentrifuge (TLA 100.4 rotor). Collect the supernatant in Epi tubes. Supernatant can be stored at this stage. Step B

Resuspend in 500 μl of solubilization buffer (50 mM KPi pH 8.0 + 400 mM NaCl + 20% glycerol) and 500 μl of 10% DDM in 1 TLA 100.4 tube Incubate in 4°C with shaking for 30min. Spin down at 80 000 rpm for 25 min in 4°C in ultracentrifuge (TLA 100.4 rotor). Collect the supernatant in Epi tubes. Supernatant can be stored at this stage. 7 – FPLC

Buffers: Buffer A 500 ml 200 ml Stock 20 mM Imidazole 10 ml 4 ml 1 M Imidazole 600 mM NaCl 60 ml 24 ml 5 M NaCl 50 mM KPi pH 8.0 25 ml 10 ml 1 M KPi pH 8.0 10% Glycerol 66.6 ml 26.6 ml 75% Glycerol 0.1% DDM 5 ml 2 ml 10% DDM Water 333.4 ml 133.4

Buffer C 500 mM Imidazole 250 ml or 17g 100 ml or 6.8g 1 M Imidazole 600 mM NaCl 60 ml 24 ml 5 M NaCl 50 mM KPi pH 7.0 25 ml 10 ml 1 M KPi pH 7.0 10% Glycerol 66.6 ml 26.6 ml 75% Glycerol 0.1% DDM 5 ml 2 ml 10% DDM Water 93.4ml 37.4 ml

Staining of SDS-PAGE gels with Coomassie Brilliant Blue

• Heat gel in staining solution and shake for 10 min.
• Poor off staining solution and add destain.
• Heat gel in destaining solution and shake.
• Replace destaining solution after 10 min and repeat until ready.

Measurements

Fermentation

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

• Autoclave closed fermenter system
• Inoculate 1.3 L LB (+100 µL [http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=A6457|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&lang=en_US%3E 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^{Kostal 2004}

• Grow ON culture of E. coli @ 30 °C
  • Use E. coli + control vector, E. coli + [http://partsregistry.org/wiki/index.php?title=Part:BBa_K190023 pArsR]-[http://partsregistry.org/Part:BBa_E1010 RFP], E. coli + [http://partsregistry.org/wiki/index.php/Part:BBa_K190032 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 h of E. coli + [http://partsregistry.org/wiki/index.php/Part:BBa_K190032 pLac-fMT]
  • Induce E. coli + [http://partsregistry.org/wiki/index.php/Part:BBa_K190032 pLac-fMT] at OD₆₀₀ ~0.6 with 0.5 mM IPTG.
• Harvest the cells @ stationary phase (after ~30 h) by spinning down @ 4000 RPM for 20 min. in Sorval centrifuge.
• Wash 2 times with TB74S buffer
• Resuspend in prewarmed (30 °C) TB74S buffer up to a OD₆₀₀ of ~25
  • Take a 1 mL sample in small aluminum boxes and dry @ 104 °C for >4 h
  • 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 1h @ 30 °C with 0μM, 10 μM, 20 μM, 50 μM and 100 μM As(III).
• For the concentration range:
  • Incubate 5 samples (of same concentration) @ 30 °C 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 demi water.
• Dry sample @ 65 °C for 2 days.
• Store @ 4 °C or -80 °C
• Determine the amount of As(III) in the cell at different stages and at different uptake concentrations using [http://en.wikipedia.org/wiki/Inductively_coupled_plasma_mass_spectrometry 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:

• Let the samples cool down.
• Dilute the samples by adding demi water up to 50 mL
• If needed, spin down 15 min. @ 4000rpm in a Sorvall centrifuge.
• Measure the arsenic concentration by [http://en.wikipedia.org/wiki/Inductively_coupled_plasma_mass_spectrometry 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 - 10 µg As/L and 0 - 100 µg As/L using a certified 1000 ppm (mg/L) stock

Fluorescence measurement

Death assay

Metal sensitivity assay^{Lewinson 2009}

Measurement:

• Grow selected strains ON in LB medium with or without antibiotic
• Induce in culture with inducer (in our case 0.5 mM IPTG)

Strains used in our tests

• Measure OD₆₀₀ of ON culture and dilute to an OD₆₀₀ of 0.05 in LB+antibiotic & inducer (IPTG in our case). Inducer should be right concentration for use in microtiterplate (because you then dilute culture 150/200=1.33 times)
• Add 150 ul of culture to 96 well microtiter plate (in triplo/quadruplo)
• Add desired concentration of selected metal in 50 μL LB+antibiotic

Metals used in our test

• Measure in Tecan Infinite 200 microplate reader (Tecan Group Ltd., Männedorf, Switzerland) or Tecan microplate *reader. Protocol:
  • Measure OD at 600 nm,
  • Every 15 minutes for 16-20hrs
  • Linear shaking, 6mm
  • 37°C

Analysis:

• Plot for different strains OD₆₀₀ against time
• Plot for different strains, the different metal concentrations against OD₆₀₀ at 12 hours

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, 15 g)
• 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

[http://openwetware.org/wiki/Ampicillin 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

[http://openwetware.org/wiki/Chloramphenicol 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

[http://openwetware.org/wiki/Kanamycin Kanamycin]

50 mg/ml Kanamycin (1000x) Stock

• 500 mg in 10 mL demi water
• 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

Destaining solution

• 16 % methanol
• 10 % acetic acid
• 74 % water

0.15 M NaCl (Saline solution, 0.9% NaCl)

• 9 g NaCl
• Dissolve in 1 L demi water

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)

[http://openwetware.org/wiki/IPTG 1 M IPTG]

• 2.38 g Isopropyl-beta-D-thiogalactopyranoside (IPTG) in 10 mL demi water.
• Filter sterilize with a 0.22 μm syringe filter.
• Store in 1 mL aliquots at -20 °C.

Sodium Arsenite (III)

• 100mM Na-As solution
• filter sterilize

Staining solution

• 0.25 % Coomassie Brilliant Blue R-250
• 50 % methanol
• 10 % acetic acid
• 40 % water

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

[http://openwetware.org/wiki/TBE 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