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Revision as of 16:20, 24 October 2010

Lab protocols

Preparation of electrocompetent E.coli cells

modified from [http://openwetware.org/wiki/Quint_Lab:electrocompetent_cells_e.coli Quint Lab]

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)

Protocol

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

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 [http://openwetware.org/wiki/Silver:_BB_Strategy Silver lab]:

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.

Silver Suffix Insertion

Silver Prefix 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)

Phusion PCR

10 µL 5x HF-Buffer
4 µL dNTPs (2.5 mM each)
1.5 µL DMSO
31.5 µL H₂O
1 µL Primer Mix
1 µL Template
1 µL Phusion DNA-Polymerase

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

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)
- These antibiotica are used 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 will 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 mRFP.

(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 ( mRFP or Luciferase)

Measuring of 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 of 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

Protocol:

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
Prepare the Luciferase Assay Reagent, by adding 10 mL of Luciferase Assay Buffer to the vial containing the Luciferase Assay Substrate
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 for the Luciferase Assay Reagent and following settings (we used a Promega GloMax®-Multi Detection System with dual injector):
- Injection volume of Luciferase Assay Reagent: 100 µL
- Delay: 20 secs
- Integration: 3 secs

Chemicals, material etc.

Used enzymes.
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

Openwetware: Quint Lab: electrocompetent cells e.coli, [http://openwetware.org/wiki/Quint_Lab:electrocompetent_cells_e.coli http://openwetware.org/wiki/Quint_Lab:electrocompetent_cells_e.coli], October 24th 2010.

Openwetware: Silver: BB Strategy, [http://openwetware.org/wiki/Silver:_BB_Strategy http://openwetware.org/wiki/Silver:_BB_Strategy], October 24th 2010.

Luciferase Assay System Technical Bulletin #TB281, Promega Cooperation, [http://www.promega.com/tbs/tb281/tb281.pdf http://www.promega.com/tbs/tb281/tb281.pdf], October 24th 2010.

@@ Line 203: / Line 203: @@
 * freeze ligation mix or continue with transformation (heatshock or electroporation)
+== Phusion PCR ==
+* 10 µL 5x HF-Buffer
+* 4 µL dNTPs (2.5 mM each)
+* 1.5 µL DMSO
+* 31.5 µL H<sub>2</sub>O
+* 1 µL Primer Mix
+* 1 µL Template
+* 1 µL Phusion DNA-Polymerase

Team:Bielefeld-Germany/Project/Protocols