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Team:Groningen/Protocols
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Protocols
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Basic Cloning Strategy:
We clone the organisms using the following strategy:
- Transform E. coli TOP10 (genotype DH 10B) with gvp (BBa_I750016), a metal ion transporter (HmtA and GlpF) and accumulation proteins.
- PCR the restriction sites out and add BioBrick pre- and suffix --> Use [http://openwetware.org/wiki/The_BioBricks_Foundation:BBFRFC10 BBFRCF10].
- Primers should be ordered for the different genes.
- Add a RBS (BBa_B0034)in the primer for the BioBrick prefix.
- Add a terminator (BBa_B0014) via cloning.
- For gvp the RBS is included in the construct, and biobrick suffix is included in the construct. The prefix is missing because of the EcoRI site in the middle of the plasmid!! This may give problems!!
- PCR restriction sites out. !!Both PCR reactions for pre/suffix and restriction sites can possibly be done in 3 seperate PCR reactions !!
- Test expression / phenotype of separate proteins (if possible in the vectors they are supplied in).
- Put both systems (gvp and metal import) on a high and low copy number (supplied by "vector group"). This is needed to prevent plasmid / expression incompatibility when both systems are used in one strain.
- The metal transporter and accumulation protein should be cloned behind each other. If possible on a synthetic operon.
- Clone the different systems for Cu, Zn, As, (Hg) in [http://www.partsregistry.org/Assembly:Rolling_assembly parallel].
- (If needed and not already supplied by "vector group") [http://www.partsregistry.org/Assembly:Rolling_assembly In parallel clone] metal sensitive promoters in front of a fluorescent protein (GFP) and in front of the gvp cluster.
- (If needed and not already supplied by "vector group") [http://www.partsregistry.org/Assembly:Rolling_assembly In parallel clone] different promoters in front of the two systems.
- Inducible like Para or Plac
- Constitutive with expected high and low expression yield
- Metal sentitive promoter (only for gvp system)
- Then try to get both systems in one E. coli strain, test different possibilities with the high + low copy nr vectors
Protocols
Cloning
[http://openwetware.org/wiki/PCR PCR]
| 12.5 μL Phusion 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]) 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
- 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 or plasmid (no death gene!), - 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 (or 800 μL when spinning cells down, see below)
- 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 Taq mastermix* 1 μL forward primer | PCR Reaction** Hotstart |
- Put PCR product on agarose gel
** Indication, actual reaction program depends on primer set (Temperature of annealing) and the length of the template (Duration of elongation)
Restriction analysis
See also Restriction, however in checking the presence of a multitude of bricks more diverse enzymes can be used. Also the incubation time can be shortened (Pour an agarose gel, wait for it to solidify and put reaction on gel) because it is not required that everything is cut.
Membrane protein isolation
- Use 20 mL of ON culture to start main culture in 1L LB medium containing 50 μg/mL ampicillin
- Incubate 37 °C, 250 RPM until OD600 = 0.6, (approximately 2.5h of incubation, check the OD600 every hour)
- Add inducer (e.g. [Team:Groningen/Protocols#IPTG|IPTG]])
- Incubate 1 h, 37 °C, 250 RPM
- Culture Wash.
- Cool culture on ice
- Spin down culture @ 8000 rpm, 10 min, 4°C
- Wash pellet with 40 ml ice-cold 50 mM KPi, pH 7.0
- Spin down culture @ 8000 rpm, 10 min, 4°C
- Resuspend pellet in 12 ml 50 mM KPi pH 7.0
- Homogenization
- Sonication on ice
- 9 cycles of 15 sec. sonication, 45 sec. rest
- Separation fractions
- Spin down @ 8000 rpm, 10 min, 4 °C
- Collect supernatant
- Spin down at 90 000 rpm, 25 min, 4 °C
- Resuspend pellet in 1 ml of 50 mM KPi pH 7.0 + 1M NaCl
- Spin down @ 80 000 rpm, 25 min, 4 °C
- Solubilization
- Resuspend in 950 μl of solubilization buffer (50 mM KPi pH 8.0 + 400 mM NaCl + 20% glycerol) and 50 μl of 10% DDM
- Incubate in 4°C with shaking for 30 min.
- Spin down @ 80 000 rpm, 25 min in 4°C
- Collect the supernatant
- (Supernatant can be stored at this stage)
- Add Ni-NTA resin (30 μL buffer A, 0.1% DDM (Bis(4-chlorophenyl)methane))
- Incubate ON @ 4 °C
- Spin down, 4 min. 3500 RPM
- Remove supernatant
- was resin with 1 mL buffer B, 0.1% DDM
- Spin down, 4 min. 3500 RPM
- Remove supernatant
- Elute protein by buffer C, 0.1% DDM (50 μL)
- Add protein loading buffer (with dithiothreitol (DTT))
- Run 12% SDS-PAGE
- Continue to Coomassie staining
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
- Dilute ON culture 1:20 in LB+Ampicillin in 50 mL greiner tube
- Incubate 37 °C, 250 RPM until an OD600 ~0.5
- Spin down 10 min. 4000 RPM, 4 °C
- Resuspend pellet in LB+Ampicillin (half the volume used before)
- Incubate @ 4 °C, 30 min.
- Load samples onto a 96-wells plate, 250 μL
- Induce by 1.25 μL of the following stock solutions
| Metal | |||||
|---|---|---|---|---|---|
| CuSO4 | 1 M | 100 mM | 10 mM | 1 mM | 0 mM |
| ZnSO4 | 1 M | 100 mM | 10 mM | 1 mM | 0 mM |
| NaAsO2 | 1 M | 100 mM | 10 mM | 1 mM | 0 mM |
| Final con. | 5000 μM | 500 μM | 50 μM | 5 μM | 0 μM |
- Measure the fluorescence and OD600 every hour.
- RFP is excited @ 580 nm and emission is measured at 609 nm.
- Store the plates between the measurements in a shaking incubator @ 37 °C.
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
- (1.5% Agar, 15 g)
- Dissolve in 1 L demi water
- Autoclave
- Store @ RT (LB) or 60 °C (LB-Agar)
TB medium
- 12 g Bacto-Tryptone
- 24 g Bacto-Yeast Extract
- 4 mL Glycerol [87%]
- Dissolve in 900ml demi water
- Separetely prepare 100 mL Kpi
- 0.17 M KH2PO4 (mw=136.09g/mol) (6.94g/300ml)
- 0.72 M 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
Buffer A
- 10 mM Imidazole
- 600 mM NaCl
- 50 mM KPi pH 8.0
- 10% Glycerol
- 0.1% DDM
- Demi water
Buffer B
- 20 mM Imidazole
- 600 mM NaCl
- 50 mM KPi pH 8.0
- 10% Glycerol
- 0.1% DDM
- Demi water
Buffer C
- 500 mM Imidazole
- 600 mM NaCl
- 50 mM KPi pH 8.0
- 10% Glycerol
- 0.1% DDM
- Demi water
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
