Team:Bielefeld-Germany/Project/Protocols
From 2010.igem.org
(→Error Prone PCR) |
(→Error Prone PCR) |
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* plate the correct colony | * plate the correct colony | ||
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Error-prone PCR –Procotols 23. Oktober 2010 | Error-prone PCR –Procotols 23. Oktober 2010 | ||
- | + | ==Directed mutagenesis – Error-prone PCR and screening system== | |
- | 1. Error PCR Step | + | ===1. Error PCR Step=== |
Mix: | Mix: | ||
- | + | * 10 µL 100 mM Tris-HCL, pH 8.3 | |
- | + | * 2.5 µL 2M KCL | |
- | + | * 3.5 µL 200 mM MgCL2 | |
- | + | * 2 µL 25 mM MnCL2 (Add immediately before initiation of the PCR) | |
- | + | * 4 µL 25 mM dCTP | |
- | + | * 4 µL 25 mM dTTP | |
- | + | * 4 µL 5 mM dATP | |
- | + | * 4 µL 5 mM dGTP | |
- | + | * 0.5 µL 100 µM 5´primer (equals 50 pmol) | |
- | + | * 0.5 µL 100 µM 3´primer (equals 50 pmol) | |
- | + | * x µL Template K389212 – total amount of 1.5 pmol | |
- | + | * 5 µL 1 U/µL Taq DNA polymerase | |
- | + | * Add Milli Q water to 100 µL total volume | |
- | + | ||
- | + | ||
- | + | * PCR program: | |
- | + | ** Start: 8 min, 98 °C (melt) | |
- | + | ** 24 cycles of: | |
- | + | *** 30 s, 98 °C (melt) | |
- | + | *** 30 s, 60 °C (anneal) | |
- | + | *** 30 s / 1 kb template, 72 °C (extension) | |
- | + | ** Final extension: 5 min, 72 °C | |
- | + | ** End: Keep at 4 °C | |
- | 2. Cloning of virA variants | + | ===2. Cloning of virA variants=== |
Clone the randomly mutated virA sequences (2.5 kB) under the control of the promoter J23110 and the RBS B0034 in the pSB1AT2 backbone. | Clone the randomly mutated virA sequences (2.5 kB) under the control of the promoter J23110 and the RBS B0034 in the pSB1AT2 backbone. | ||
(See above for protocol of Prefix- / Suffix-Insertion) | (See above for protocol of Prefix- / Suffix-Insertion) | ||
- | 3. | + | ===3. Primary selection of novel virA variants=== |
Transform the libraries of virA variants into electro-competent E. coli, in which K389101 and thereby the read out system via kanamycin resistanc is already present | Transform the libraries of virA variants into electro-competent E. coli, in which K389101 and thereby the read out system via kanamycin resistanc is already present | ||
(See above for protocol of transformation) | (See above for protocol of transformation) | ||
Plate the transformed bacteria on selective LB-Agar. When producing the plates add the following mixture of substances to the hand-warm LB-Agar: | Plate the transformed bacteria on selective LB-Agar. When producing the plates add the following mixture of substances to the hand-warm LB-Agar: | ||
- | + | * 1 mL Ampicillin stock solution (final concentration 100 µg / mL) | |
- | + | * 286 µL Chloramphenicol stock solution (final concentration 10 µg / mL) | |
- | + | ** Both to ensure stability of both plasmids | |
- | + | * 50 mL Substance Mix – including capsaicin, dopamine, homovanillic acid and 3-O methyldopamine (all final concentration of 200 µM) | |
- | + | ** Any bacteria with a virA variation that is activated by at least one of these novel inductors will express the kanamycin resistance | |
- | + | * 2 mL Kanamycin stock solution (final: 100 µg / mL) | |
- | + | ** Compareable high kanamycin concentration to allow only these bacteria to grow which show a strong expression o the kanamycin resistance. (This optimal concentration was previously determined in MIC analysis.) | |
Incubate plates upside down at 37 °C for 16 – 20 h. | Incubate plates upside down at 37 °C for 16 – 20 h. | ||
The grown colonies must include a virA variant that is either constitutively active or had been induced by at least one of the tested substances. | The grown colonies must include a virA variant that is either constitutively active or had been induced by at least one of the tested substances. | ||
- | 4. Quantitative analysis of virA variants after induction with novel substances | + | ===4. Quantitative analysis of virA variants after induction with novel substances=== |
To quantify the induction profile of the selected virA variants it is appropriate to change the read out system from kanamycin resistence to luciferase expression. This complex and time consuming task can easily be achieved without any cloning step, when using the advantage of our two plasmid system. | To quantify the induction profile of the selected virA variants it is appropriate to change the read out system from kanamycin resistence to luciferase expression. This complex and time consuming task can easily be achieved without any cloning step, when using the advantage of our two plasmid system. | ||
- | + | * Pick a colony of interest and plate it on a fresh LB-Agar, containing Amp (100 µg / mL) and Cm (10 µg / mL) | |
- | + | * Incubate upside down at 37 °C for 16 – 20 h. | |
- | + | * Isolate plasmids from the bacterial lawn with a miniprep | |
- | + | ** In this step two plasmids are isolated – One includes the virA variants in a normal pSB1AT3 backbone. The second type with the kanamycin read out has the special r6k ori, which can only amplify in certain E. coli strains. | |
- | + | * Transform the isolated plasmids to a strain which is not capable to amplify the r6k ori (e.g. TOP10) | |
- | + | * Plate the transformants on LB-Agar with Amp (100 µg / mL) and isolate plasmids from the bacterial lawn with a miniprep | |
- | + | ** Here you are only harvesting the plasmid including the virA variant in psB1AT3, since the other plasmid was not amplified in the grown E. coli. | |
- | + | * Transform the plasmid with the virA variant into competent cells, already including a second plasmid with a read out system luciferase or RFP. | |
- | + | * Grow the transformants in shake flaks in LB-Media containing ampicillin (100 µg / mL), chloramphenicol (10 µg / mL) and 200 µM of one testing substance (e.g. capsaicin). As a control you should also grow the same bacteria without any inductor and with the native inductor acetosyringone. | |
- | + | * Take samples in certain intervals (e.g. 1 h) and measure the activity of the luciferase or RFP (you might store the samples at -80 °C before you measure the activity) | |
== Restriction analysis == | == Restriction analysis == |
Revision as of 12:38, 24 October 2010
Lab protocols
Preparation of electrocompetent E.coli cells
modified from Quint Lab
http://openwetware.org/wiki/Quint_Lab:electrocompetent_cells_e.coli
Materials: • 10 mL LB-Medium (1% Bacto Trypton; 0,5 % Yeast Extract; 0,5% NaCl) • 2L cooled bidest H2O • 200 mL cooled, sterile-filtered 10% Glycerol • box with ice-water for 2-litre-flask • 4 pre-cooled 250 mL (or 2x500 mL) bins for centrifugation • 2 pre-cooled 50 mL Falcons • centrifuge pre-cooled to 2°C (max. 4°C)
Method
- inoculate 2x10 mL LB with bacterial stock; incubate over night at 37°C and 200 rpm
- inoculate 2x250 mL LB in 1-litre-flask with OD600=0,1 @37°C or @ 19°C over night
- incubate until OD600 0,4-0,6 (~5 h)
- from now on everything is done at 2-4°C (best in a cold room)
- cool 1L-culture 10-15 minutes in ice water (shake sometimes)
- divide culture into 2x5 cooled 50 mL Falcon-tubes for centrifugation
- centrifuge 10min @ 4°C, 4000 rcf
- discard supernatant
- resuspend pellet in 5 mL bidest H2O
- add bidest H2O up to 50 mL
- centrifuge 10min @ 4°C, 4000 rcf
- discard supernatant immediately
- resuspend pellet in residual supernatant
- add bidest H2O up to 50 mL
- centrifuge 10min @ 4°C, 4000 rcf
- discard supernatant immediately
- resuspend pellet in residual supernatant
- transfer suspension in 2x50 mL Falcons
- add 10% Glycerol up to 50 mL
- centrifuge 5 min @ 4°C, 4000 rcf
- discard supernatant
- estimate volume of the pellet; fill up with equal volume of 10% Glycerol
- resuspend pellet on ice; ‘’’don´t vortex!!’’’ (just shake cautiously)
- divide cells into 150 µL Alicuots (use 1,5 mL Eppis)
- freeze in liquid N2 or dry-ice
- store @ -80°C
Preparation of heat shock competent E.coli cells
Method
- inoculate 2x5 mL LB with bacterial stock; incubate over night at 37°C and 200 rpm
- inoculate 2x250 mL SOB in 1-litre-flasks with OD600=0,1
- incubate @19°C until OD600=0,4-0,6 (20h)
- from now on everything is done at 2-4°C (best in a cold room)
- cool 1L-culture 10-15 minutes in ice water (shake sometimes)
- divide culture into 2x5 cooled 50 mL Falcon-tubes for centrifugation
- centrifuge 10min @ 4°C, 4000 rcf
- discard supernatant
- resuspend cells in 80 mL TB buffer
- cool cells for 10 min on ice
- centrifuge 10min @ 4°C, 2500 rcf
- discard supernatant
- resuspend cells in 20 mL TB buffer
- add 1,5 mL Dimethyl sulfoxide (DMSO) for each 20 mL cells (DMSO concentration = 7%)
- cool cells for 10 min on ice
- divide cells into 150 µL Alicuots (use 1,5 mL Eppis)
- freeze in liquid N2 or dry-ice
- store @ -80°C
Transformation via electroporation
- Thaw 50 µL competent E.coli cells on ice, dilute with icecold 50µL Glycerin (10%) if necessary
- Add 0,5-5 µL plasmid to 50 µl electrocompetent cells
- Store cells on ice for 1 minute
- Electroporate at U= 2,5 kV, C= 25 µF, R = 200 Ώ
- Transfer transformation reaction to 450 µL SOC-Medium and shake 1 h at 37°C
- Centrifuge 2 min @ 800 rpm and plate on selective LB-Medium
Heat shock transformation
- Thaw 150 µL competent E. coli cells on ice
- add max. 10 µL DNA (the less the better your transformation works but at least about 50 ng vector)
- incubate 30 min on ice
- heatshock: 42 °C, 45 s (water bath because of quick heat transfer)
- 1 min on ice
- add 1 mL prewarmed SOC-medium
- incubate: 45 - 60 min, 37 °C
- plate 100 µL
- spin down the remaining cells (2 min, 5000 g), discard most of the supernatant, resuspend the cells in the remaining medium and plate them
Silver BioBrick Assembly
modified from Silver lab: http://openwetware.org/wiki/Silver:_BB_Strategy
This assembly method can be used for BioBricks which are bigger than 150 bp. The BioBrick should be at least 500 bp bigger or smaller than the backbone. The BioBrick, which complies with these conditions, is used as the insert and is assembled into the prefix or suffix of the other used BioBrick, called vector. So you have to differentiate between a prefix and a suffix insertion.
Suffix Insertion
- Digestion of insert: at least 700 ng DNA / 10 µL volume, 1 µL 10x Tango buffer, 0.5 µL XbaI, 1 µL PstI. Digest for 2 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel try to avoid staining or exposure to ultraviolet light of the insert.
- Digestion of vector about 700 ng DNA / 10 µL volume, 1 µL 10x orange buffer, 0.5 µL SpeI, 0.5 µL PstI. Digest for 2h at 37 °C, afterwards inactivation for 20 min at 80 °C. Add 1 µL SAP (shrimp alcaline phosphatase) and 1.2 µL 10 x SAP buffer, incubate for 1 h at 37 °C. Clean up the vector with a PCR clean-up kit.
- Ligation: after digestion and clean-up: 50 - 200 ng of vector, 3 - 10 fold molar access of insert, 20 µL ligation volume, 2 µL T4-Ligase-Buffer, 1 µL T4-Ligase. Incubate for 1 h at 37 °C, afterwards inactivation for 5 min at 70 °C. Then: store at -20 °C or transform.
Prefix Insertion
- Digestion of insert: at least 700 ng DNA / 10 µL volume, 1 µL 10x BamHI buffer, 0.5 µL EcoRI, 0.5 µL SpeI. Digest for 2 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel try to avoid staining or exposure to ultraviolet light of the insert.
- Digestion of vector about 700 ng DNA / 10 µL volume, 1 µL 10 x Tango buffer, 0.5 µL EcoRI, 0.5 µL XbaI. Digest for 2h at 37 °C, afterwards inactivation for 20 min at 80 °C. Add 1 µL SAP (shrimp alcaline phosphatase) and 1.2 µL 10 x SAP buffer, incubate for 1 h at 37 °C. Clean up the vector with a PCR clean-up kit.
- Ligation: after digestion and clean-up: 50 - 200 ng of vector, 3 - 10 fold molar access of insert, 20 µL ligation volume, 2 µL T4-Ligase-Buffer, 1 µL T4-Ligase. Incubate for 1 h at 37 °C, afterwards inactivation for 5 min at 70 °C. Then: store at -20 °C or transform.
Variations
- A digestion over night is possible. If you digest over night use only 0.1 µL restriction enzyme.
- It is also possible to use PCR product as insert. Digest after PCR with corresponding restriction enzymes and clean up with PCR clean-up kit. This could lead to higher yields of insert DNA because a lot of DNA gets lost during the gel electrophoresis clean up.
- Sometimes some BioBricks are hard to assemble. Then you have to clean up the vector by gel electrophoresis as well.
3A assembly
modified from: ...
Digestion
- Thaw DNA from upstream and downstream part and the destination plasmid on ice.
- destination plasmid has to carry the ccdB gene <partinfo>P1010</partinfo> as insert and has to have a different antibiotic resistance than the plasmids carrying the upstream and downstream parts
- DNA has to be cleaned (by MiniPrep or after a PCR)
- 500 ng DNA / digestion mix for upstream and downstream part, 150 ng DNA / digestion mix for destination plasmid (total volume of mix 10 µl, dilute with ddH20 if necessary)
- add 1 µl of buffer and restriction enzymes as shown in the following table:
Upstream part | Downstream part | Destination plasmid | |
---|---|---|---|
enzyme 1 | 1 µl EcoRI | 0.5 µl XbaI | 0.5 µl EcoRI |
enzyme 2 | 1 µl SpeI | 1 µl PstI | 0.5 µl PstI |
buffer | BamHI | Tango | Orange |
- incubation of the digestion mixes for 2 h at 37 °C, afterwards heat inactivation for 20 min at 80 °C
- continue with ligation or freeze the mixes
Ligation
- ligation mix:
- 2 µl ddH2O
- 5 µl of every digestion mix (so 15 µl in total)
- 2 µl T4-DNA-ligase buffer (thaw on ice!)
- 1 µl T4-DNA-ligase
- incubate 1 h at 37 °C, afterwards heat inactivation for 5 min at 70 °C
- freeze ligation mix or continue with transformation (heatshock or electroporation)
Colony PCR
- pick one colony with a sterile tip and elute it in 100 µl ddH20 or medium
- store the colony in 4°C while colony PCR is running
- one reaction mix contains:
- 2.5 µl 10x buffer
- 0.75 µl MgCl2
- 1 µl dNTPs
- 0.5 µl primer mix (prefix/suffix primers or sequencing primers)
- 19.25 µl ddH2O
- 0.5 µl taq-polymerase
- 0.5 µl template
- PCR program:
- start: 8 min, 98 °C
- 30 cycles of:
- 30 s, 98 °C
- 30 s, 60 °C
- 30 s / 1 kb template, 72 °C
- finish: 5 min, 72 °C
- gel electrophoresis: check the fragment size
- plate the correct colony
Error-prone PCR –Procotols 23. Oktober 2010
Directed mutagenesis – Error-prone PCR and screening system
1. Error PCR Step
Mix:
- 10 µL 100 mM Tris-HCL, pH 8.3
- 2.5 µL 2M KCL
- 3.5 µL 200 mM MgCL2
- 2 µL 25 mM MnCL2 (Add immediately before initiation of the PCR)
- 4 µL 25 mM dCTP
- 4 µL 25 mM dTTP
- 4 µL 5 mM dATP
- 4 µL 5 mM dGTP
- 0.5 µL 100 µM 5´primer (equals 50 pmol)
- 0.5 µL 100 µM 3´primer (equals 50 pmol)
- x µL Template K389212 – total amount of 1.5 pmol
- 5 µL 1 U/µL Taq DNA polymerase
- Add Milli Q water to 100 µL total volume
- PCR program:
- Start: 8 min, 98 °C (melt)
- 24 cycles of:
- 30 s, 98 °C (melt)
- 30 s, 60 °C (anneal)
- 30 s / 1 kb template, 72 °C (extension)
- Final extension: 5 min, 72 °C
- End: Keep at 4 °C
2. Cloning of virA variants
Clone the randomly mutated virA sequences (2.5 kB) under the control of the promoter J23110 and the RBS B0034 in the pSB1AT2 backbone. (See above for protocol of Prefix- / Suffix-Insertion)
3. Primary selection of novel virA variants
Transform the libraries of virA variants into electro-competent E. coli, in which K389101 and thereby the read out system via kanamycin resistanc is already present (See above for protocol of transformation) Plate the transformed bacteria on selective LB-Agar. When producing the plates add the following mixture of substances to the hand-warm LB-Agar:
- 1 mL Ampicillin stock solution (final concentration 100 µg / mL)
- 286 µL Chloramphenicol stock solution (final concentration 10 µg / mL)
- Both to ensure stability of both plasmids
- 50 mL Substance Mix – including capsaicin, dopamine, homovanillic acid and 3-O methyldopamine (all final concentration of 200 µM)
- Any bacteria with a virA variation that is activated by at least one of these novel inductors will express the kanamycin resistance
- 2 mL Kanamycin stock solution (final: 100 µg / mL)
- Compareable high kanamycin concentration to allow only these bacteria to grow which show a strong expression o the kanamycin resistance. (This optimal concentration was previously determined in MIC analysis.)
Incubate plates upside down at 37 °C for 16 – 20 h. The grown colonies must include a virA variant that is either constitutively active or had been induced by at least one of the tested substances.
4. Quantitative analysis of virA variants after induction with novel substances
To quantify the induction profile of the selected virA variants it is appropriate to change the read out system from kanamycin resistence to luciferase expression. This complex and time consuming task can easily be achieved without any cloning step, when using the advantage of our two plasmid system.
- Pick a colony of interest and plate it on a fresh LB-Agar, containing Amp (100 µg / mL) and Cm (10 µg / mL)
- Incubate upside down at 37 °C for 16 – 20 h.
- Isolate plasmids from the bacterial lawn with a miniprep
- In this step two plasmids are isolated – One includes the virA variants in a normal pSB1AT3 backbone. The second type with the kanamycin read out has the special r6k ori, which can only amplify in certain E. coli strains.
- Transform the isolated plasmids to a strain which is not capable to amplify the r6k ori (e.g. TOP10)
- Plate the transformants on LB-Agar with Amp (100 µg / mL) and isolate plasmids from the bacterial lawn with a miniprep
- Here you are only harvesting the plasmid including the virA variant in psB1AT3, since the other plasmid was not amplified in the grown E. coli.
- Transform the plasmid with the virA variant into competent cells, already including a second plasmid with a read out system luciferase or RFP.
- Grow the transformants in shake flaks in LB-Media containing ampicillin (100 µg / mL), chloramphenicol (10 µg / mL) and 200 µM of one testing substance (e.g. capsaicin). As a control you should also grow the same bacteria without any inductor and with the native inductor acetosyringone.
- Take samples in certain intervals (e.g. 1 h) and measure the activity of the luciferase or RFP (you might store the samples at -80 °C before you measure the activity)
Restriction analysis
- Digest BioBrick of interest: about 400 ng DNA / 10 µL volume, 1 µL 10x orange buffer, 0.5 µL NotI or PstI. Digest for 2 h at 37 °C. NotI is used to determine the length of the BioBrick and the plasmid backbone, PstI ist used to determine the length of the BioBrick in the plasmid backbone.
- Gel electrophoresis: add 2 µL loading buffer to every digestion mix, apply about 100 - 200 ng DNA / pocket in gel. Don't forget to apply the uncut BioBrick as well. A good agarose concentration for BioBricks between 0.2 and 3 kb is 1.5 %. The smaller your BioBrick of interest is the higher the agarose concentration should be and vice versa. The gel electrophoresis is made with TAE-buffer. Be sure that you melt your agarose gel in the same buffer you use for the electrophoresis later.
Cultivation for measuring mRFP and Luciferase expression
- Inoculate 10 mL LB containing desired Antibiotic with glyccerol stock
- Cultivate over night at 37 °C and 175 rpm
- Measure the OD600
- Prepare shake flasks with LB, antibiotic and inductor
- For Luciferase Measurement at least 10 mL starting volume
- For mRFP Measurement at least 20 mL starting volume
- Inoculate the main culture with a starting OD600 of 0.1
- Cultivate at 37 °C and 175 rpm
- Take a sample at least every hour and measure the OD600
- The sample handling depends on the readout strategy and is described in the corresponding measuring protocol
Measuring mRFP
- Take at least 500 µL sample for each measurement (200 µL is needed for one measurement) so you can perform a repeat determination
- freeze samples at -80 °C for storage
- to measure the samples unthaw at room temperature and fill 200 µM of each sample in one well of a black, flat bottom 96 well microtiter plate (perform at least a repeat determination)
- measure the Fluorescence in a platereader (we used a Tecan Infinite® m200platereader ) with following settings
- 20 sec orbital shaking (1 mm amplitude with a frequenzy of 87.6 rpm)
- Measurement mode: Top
- Excitation: 584 nm
- Emission: 620 nm
- Number of reads: 25
- Manual gain: 150
- Integration time: 20 µs
Measuring Luciferase
- For the luciferase detection we used a [http://www.promega.com/tbs/tb281/tb281.pdf Promega Luciferase Assay System], containing a Cell Culture Lysis Reagent, Luciferase Assay Substrate and Luciferase Assay Buffer
- Prepare reaction tubes with 10 µL of high salt buffer (1M K2HPO4, 20mM EDTA, pH 7.8)
- Add 90 µL sample, mix and freeze at -80 °C
- For the measurement unfreeze by placing the tubes in room temperature water
- Add 300 µL of freshly prepared lysis mix ( 1X Cell Culture Lysis Reagent, 1,25 mg/mL lysozyme, 2,5 mg/mL BSA, add Water for desired Volume)
- Mix and incubate the cells for 10 minutes at room temperature
- Fill each well of a white, flat bottom 96 well microtiter plate with 20 µL of cell lysate
- For the Detection of Luciferase use a plate reading luminometer with Injector and following settings (we used a Promega GloMax®-Multi Detection System with dual injector)
- blank
Chemicals, material etc.
Enzyme | Producer |
---|---|
Pfu DNA-polymerase | Promega |
Phusion DNA-polymerase | Finnzymes |
Restriction enzymes | Fermentas |
Shrimp alcaline phosphatase | Fermentas |
T4-DNA-Ligase | Fermentas |
taq-DNA-polymerase | Bioline |
TAE buffer
For 1 L of 50 x TAE buffer you need:
- 242.48 g Tris
- 41.02 g Sodiumacetate
- 18.612 g EDTA
- adjust pH to 7.8
- solve in dH2O
20 mL of the stock is diluted in 1 L dH2O for the gel electrophoresis.
DNA loading buffer
- 50 % (v/v) Glycerine
- 1 mM EDTA
- 0.1 % (w/v) Bromphenol blue
- solve in ddH20
LB medium
For 1 L of LB medium you need:
- 10 g Trypton
- 5 g yeast extract
- 10 g NaCl
- 12 g Agar-Agar (for plates)
- adjust pH to 7.0
Cell Culture Lysis Reagent
- 25 mM Tris-phosphate (pH 7.8)
- 2 mM DTT
- 2 mM 1,2-diaminocyclohexane-N,N,N´,N´-tetraacetic acid
- 10 % glycerol
- 1 % Triton® X-100
References
Technical Bulletin for Promega Cooperation Luciferase Assay System [http://www.promega.com/tbs/tb281/tb281.pdf http://www.promega.com/tbs/tb281/tb281.pdf], October 24th 2010.