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
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 ([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
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 [http://www.fermentas.com/catalog/re/ecori.htm EcoRI] (Non-fast digest)
- 1 μL [http://www.fermentas.com/catalog/re/bcui.htm 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 [http://partsregistry.org/Part:BBa_P1010:Design 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:
- 3 μL 100 µM 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)
- (ANTI-SENSE) 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.
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 [http://partsregistry.org/Part:pSB1AC3 pSB1AC3] or [http://partsregistry.org/Part:pSB3K3 pSB3K3] carrying ccdB deathgene
- Incubate 1 h @ 37 °C, 200 RPM
- 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 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: [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
- 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 [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 (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
[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
- 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
[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
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