"
Team:Groningen/Protocols
From 2009.igem.org
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
|
Protocols
|
|---|
Protocols
Cloning
[http://openwetware.org/wiki/PCR PCR]
| 21 μL mastermix* 1 μL forward primer | PCR Reaction Hotstart |
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])
- 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] or [http://www.zymoresearch.com/content/zymoclean-gel-dna-recovery-kit-d4001-d4002-d4007-d4007-d4001s Zymoclean™ Gel DNA Recovery Kit]) 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
- 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.
- Cells are divided in 50 μL aliquots
- 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
| 21 μL mastermix* 1 μL forward primer | PCR Reaction Hotstart |
- 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. coliKostal 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:
| Stage 1 | Stage 2 | |
| Power(max) | 1200 | 1200 |
| Power(%) | 100 | 100 |
| Ramp(min) | 15 | 15 |
| Hold(min) | 0 | 30 |
| Temp(°C) | 140 | 210 |
- 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 assayLewinson 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
| Test 1: | Test 2: |
|---|---|
| WT (+pSB1AC3) | WT (+pSB1AC3) |
| pLac-HmtA | pLac-HmtA |
| pLac-GlpF | pLac-GlpF |
| pLac-GlpF-fMT | pLac-GlpF-fMT |
| plow-GlpF-fMT | |
| pLac-GlpF |
- 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
| Metal Concentration | ||||
|---|---|---|---|---|
| NaAsO2 | 0 μM | 1 μM | 10 μM | 50 μM |
| CuSO4 | 0 μM | 50 μM | 250 μM | 500 μM |
- 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)
IPTG
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
