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From 2010.igem.org

Contents 1 Lab protocols 1.1 Preparation of electrocompetent E.coli cells 1.2 Preparation of heat shock competent E.coli cells 1.3 Transformation via electroporation 1.4 Heat shock transformation 1.5 Silver BioBrick Assembly 1.5.1 Suffix Insertion 1.5.2 Prefix Insertion 1.5.3 Variations 1.6 3A assembly 1.6.1 Digestion 1.6.2 Ligation 1.7 Colony PCR 1.8 Error Prone PCR 1.9 Restriction analysis 1.10 Cultivation for measuring mRFP and Luciferase expression 1.11 Measuring mRFP 1.12 Measuring Luciferase 1.13 Chemicals, material etc. 1.13.1 TAE buffer 1.13.2 DNA loading buffer 1.13.3 LB medium 1.13.4 Cell Culture Lysis Reagent 2 References

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

1. Error PCR Step

• 10 µL Tris-HCL (100mM)
• 2,5 µL KCL (2M)
• 3,5 µL MgCL2 (200mM)
• 4 µL Error DNTPs (2,5mM)
• 1 µL Primermix
• x µL Template
• x µL H20

• 2L MnCL2 (25mM)

• 5µL Taq

----

100 µL

• PCR program:
  • start: 8 min, 98 °C
  • 24 cycles of:
    • 30 s, 98 °C
    • 30 s, 60 °C
    • 30 s / 1 kb template, 72 °C
  • finish: 5 min, 72 °C

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.

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

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.