Team:EPF-Lausanne/Notebook

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Latest revision as of 23:27, 29 October 2008

Notebook

Protocols

Reference : Most of these protocols come from http://openwetware.org

Media and buffers

LB : Luria Browth medium

We used a dehydrated culture medium from [http://www.condalab.com/pdf/1551.pdf Pronadisa] containing :

• Triptone 10.0 g/L
• Yeast Extract 5.0 g/L
• Sodium chloride 10.0 g/L

Dissolve 25g of the mix in 1L of distilled water. For plates pouring, add 15g agar for 1 L of solution. Shake and close, then autoclave it to sterilize.

SOC

Super Original Catabolite Repressor

• Bactotryptone 20g.
• Bacto-yeast extract 5g.
• NaCl 0.5g.
• 1M KCl 2.5ml
• ddH2O to 1000 ml
• Total Volume 1000ml

Adjust pH to 7, with 10N NaOH. Autoclave to sterilize Add 20 ml of 1M glucose before use.

TE

10xTE for 1 liter from stock solutions 10 ml 1M Tris-HCl pH 8.0 2 ml 0.5M EDTA pH 8.0 988 ml ddH2O

→ 10xTE is 10 mM Tris-HCl and 1 mM EDTA

• For the Tris-HCl use Tris base and adjust to desired pH using HCl.

SOB: Super Original Broth

Used in growing bacteria for preparing chemically competent cells Ingredients

• 0.5% (w/v) yeast extract
• 2% (w/v) tryptone
• 10 mM NaCl
• 2.5 mM KCl
• 20 mM MgSO4

Per liter:

• 5 g yeast extract
• 20 g tryptone
• 0.584 g NaCl
• 0.186 g KCl
• 2.4 g MgSO4

!!! Adjust to pH 7.5 prior to use. This requires approximately 25 ml of 1M NaOH per liter.

CCMB80 buffer

For 1L

• 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)
• 80 mM CaCl2.2H2O (11.8 g/L)
• 20 mM MnCl2.4H2O (4.0 g/L)
• 10 mM MgCl2.6H2O (2.0 g/L)
• 10% glycerol (100 ml/L)
• adjust pH DOWN to 6.4 with 0.1N HCl if necessary
  • adjusting pH up will precipitate manganese dioxide from Mn containing solutions.
• sterile filter and store at 4°C
• slight dark precipitate appears not to affect its function

Antibiotics

For each antibiotic, solutions of 5 ml have been done and stored at -20°C (09.07.08)

Cloning

Competent cells preparation

We followed the protocol coming from [http://openwetware.org/wiki/TOP10_chemically_competent_cells OpenWetWare].

Transformation

1. Thaw competent cells on ice for 30 minutes.
2. Mix cells and DNA, put back on ice for 20 minutes.
3. Heat shock: 45 seconds at 42° C.
4. Add 200 μl of LB broth (without antibiotics).
5. Incubate at 37° C, 230 rpm for 1 hour.
6. Plate the cell culture on agar containing the correct antibiotic.
7. Incubate at 37° C overnight.

Plasmid Purification

We use the [http://www1.qiagen.com/HB/QIAprepMiniprepKit_EN QIAprep Miniprep Spin Kit].

• To get a higher DNA concentration, use only 30 ml Elution Buffer.

Ligation

Preparation:

1. Digest insert and vector.
2. Example:
   • 700 ng DNA
   • 1 μl NEB Buffer 2 (or depending on enzymes used)
   • 1 μl BSA 10X Buffer
   • 0.1 μl Enzyme 1
   • 0.1 μl Enzyme 2
   • Complete with water to final volume of 10 μl.
   • Incubate at 37° C for 1h30 - 2h.
   • If DNA concentration is low, complete to e.g. 20 μl instead and adjust Buffer volumes accordingly.
   • After digestion, treat the vector with Antarctic Phosphatase (1 μl AP, 1 μl AP Buffer) to prevent autoligation. Incubate for 20 minutes at 37° C.
3. Run digested sample on an agarose gel.
4. Purify the fragments of interest using a Gel Extraction Kit.

Ligation:

• For correct reaction calculations, follow [http://openwetware.org/wiki/DNA_ligation DNA Ligation Protocol on OWW]
• The following simplified protocol worked for inserts up to a couple of hundreds basepairs:
  • 6 μl Insert
  • 1.5 μl Vector
  • 1 μl Ligase Buffer with ATP
  • 0.5 μl T4 DNA Ligase
  • 1 μl Water
• Autoligation Control: Prepare the same reaction again but replace Insert with water.
• Incubate 30 minutes at room temperature.

MITOMI

Target DNA Protocol

Materials:

5’Comp Cy5 primer

• 5’Cy5-GTC ATA CCG CCG GA
• order from IDT at 1umole, HPLC purified
• suspend to 500uM

Library primers:

• See spreadsheet for details
• 5’ Linker - BINDING SITE – 3’ Linker – TCCGGCGGTATGAC
• 5’ Linker generally: AAC
• Binding Site Generally a 12mer but can be longer
• 3’Linker generally: C
• order from IDT in a 96 well v-bottom plate suspended at 150uM in TE

Klenow Fragment 3’-5’ exo-

• Order from NEB (Cat #: M0212L)

dNTPs at 10mM each

• order from Roche (PCR Nucleotide Mix: 11581295001)

Method:

Synthesis reaction: 3uL dNTP 3uL Buffer 2 2uL Library primer 0.4uL 5’Comp Cy5 primer 1uL Klenow 3’-5’ exo- 20.6uL dH2O 30uL Final Volume

Prepare a master mix without the library primer, and load 28uL into a 96 well plate. Add 2uL of Library primer to the 96 well plate. Cycle as follows:

• 37°C for 1 hour
• 75°C for 20 mins
• ramp down to 30°C at 0.1°C/sec
• hold at 4°C

After the synthesis add 70uL of 0.5% BSA in dH2O to each well and transfer to a 384 well plate (flat bottom). Prepare dilutions as needed using 0.5% BSA dH2O.

To set up the 384 load 30uL 0.5% BSA dH2O into all columns except 1, 7, 13, 19.

Transfer the column 1 of the klenow reaction (100uL total) to column 1 row A (A1) on the 384 well plate. Perform 5 dilutions of 70uL sample into the adjacent wells containing 30uL BSA dH2O. After the 5th dilution (in column 6) discard the remaining 70uL.

Transfer column 2 of the klenow plate into A7 and perform dilutions.

Column3 -> A13, column 4 -> A19, Column 5->B1, col 6-> B7, col7->B13, col8 -> B19

You should have 30uL of target DNA in each well of the 384 plate now. Add 30uL of 0.5% BSA dH2O to each well.

The samples are now ready to be spotted.

2-Step PCR Reaction

Purpose: generate linear PCR templates for in vitro transcription/translation of cDNA clones using E.coli colonies as starting material. Should also be able to amplify genomic targets.

Template: E.coli colonies picked from agar plates Primer stock: 5’ + 3’ gene specific primers each at 500uM Primers pair: 5’+3’ gene specific primers 1/10 in dH2O to 50uM each Polymerase: HiFi Plus (Roche)

• suspend colony in 2.5uL lyse ‘n go buffer (Pierce)
• optionally 2.5uL of an overnight LB culture can also be used (adjust dH2O accordingly)
• heat to 95°C for 7 minutes then cool to 4°C
• add 1uL primer pair and 46.5 uL PCR Mix

PCR Mix: 1uL dNTP 10uL 5x Buffer + MgCl2 .5uL HiFi DNA polymerase 35uL dH2O 46.5uL Final Volume

Temp. cycles: 1 94°C 4:00 2 94°C 0:30 3 55°C 1:00 4 72°C 2:00 5 Cycle 2-4 30x 6 72°C 7:00 7 4°C forever

• run 1uL of product on gel to check for presence and length
• optionally: run a Qiagen PCR purification

2nd step of PCR

• 5’ext1 and 3’ext2 each at 50uM
• Primer Mix: 5’ext1 + 3’ext2 primers diluted 1/200 in dH2O to 250nM each
• Template = previous PCR mix

1uL dNTP 10uL 5x Buffer + MgCl2 1uL Primer Mix 0.5uL template 0.5uL HiFi Plus DNAp 37uL dH2O 50uL Final Volume

Temp. cycles: 1 94°C 4:00 2 94°C 0:30 3 55°C 1:00 4 72°C 2:00 5 Cycle 2-4 10x 6 72°C 7:00 7 4°C forever

• 5’Final and 3’Final at 500uM
• Final Primer Mix: 5’Final and 3’Final diluted 1/10 in dH2O
• add 1uL of Final Primer Mix

Temp. cycles: 1 94°C 4:00 2 94°C 0:30 3 50°C 1:00 4 72°C 2:00 5 Cycle 2-4 25-30x 6 72°C 7:00 7 4°C forever

• check 1uL product on a 1% agarose gel
• no further purification is necessary even though PCR purification is recommended, eluting in 50uL of elution buffer (EB) or pH adjusted dH2O

In vitro transcription translation (ITT)

Linear PCR templates are transcribed and translated using the RTS 100 E.coli HY Kit (ROCHE) Master Mix:

• 12uL/sample E.coli lysate
• 10uL/sample Reaction mix
• 12uL/sample Amino Acid
• 1uL/sample Methionine
• 5uL/sample reconstitution buffer
• 6uL ddH2O
• 2uL Linear PCR template
• 2uL GFP labelled tRNA

Incubate for 4-6h at 30°C

On chip experiment

Material:

• MITOMI device bonded to epoxy slide with spotted target DNA array
• set of pressure valve, tubing, manometer and pressure gauge
• Fluorescence scanner
• ITT product (transcription factor)
• synthetic AHLs solution (stock 1mM)

Method:

• Load control lines with dH2O at 5psi
• Run 2mg/ml BSA-biotin at 5psi for 20min, chambers closed
• Run 500ug/ml NA/PBS for 20min
• Run PBS for 5min
• Close button, continue PBS for 1-2min
• Run 2mg/ml BSA-biotin for 20min
• Run PBS for 10min
• Run 1/500 penta-His Antibody in PBS for 5min
• Open button, continue antibody for 20min
• Run PBS for 2min
• Run ITT product with corresponding AHL for 20min (50ul ITT, 10ul of 1:100 diluted AHL stock)
• Close sandwich, open chamber, incubate at RT for 90min
• Close button, close chamber, open sandwich and run PBS for 5min
• Scan for red and green fluorescence

Parts Characterization

BBa_K092600 Characterization:

• Three 5 ml cultures of LB medium and antibiotic (ampicillin, 20 µg/ml) were inoculated with single colonies from a glycerol stock stored at –80°C.
• Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm.
• Cultures were diluted 1:1000 into 5 ml of fresh medium and grown to an OD600 of 0.2 under the same conditions as before.
• Twenty-four 100 µl aliquots of each of the cultures were transferred into a flat-bottomed 96 well plate (BD-96).
• Tetracycline stock solution was prepared at a concentration of 6 mg/ml.
• Varying quantities of tetracycline was added to each well to yield 14 different final concentrations. Three replicate wells were measured for each concentration of tetracycline. Three wells were each filled with 200 µl of culture medium to measure the background.
• Plate was left in incubator for 4 hours at 37°C at 60rpm.
• The plate was incubated in a multi-well fluorimeter (Perkin Elmer) at 37°C and assayed with a protocol of fluorescene measurements at 530/580 excitation/emission wavelength filters.
• The transfer function fluorescence intensity after four hours of expression. Error bars representing the 95% confidence interval in the population for the independent samples.
• Flow cytometry was carried out with samples to check for presence of RFP producing cells.

Microfluidics

SU-8 wafers

Material:

• 100mm silicon wafer
• SU-8 negative photoresist - GM1060 from [http://www.gersteltech.ch Gersteltech]

Method:

• Oxygen plasma
• dispose GM1060 on wafer and spin coat at 2000 rpm for 40s.
• Soft-bake for 5min at 130°C with a slow temperature increase/decrease (ramp of 4C°/min).
• Expose using corresponding mask (Control mask) for 16s. at 275mW.
• Post-exposure bake for 30min at 100°C with a slow temperature increase/decrease (ramp of 4C°/min).
• Develop in PGMEA bath for 2x5min and rinse with 2-propanol
• Air dry and inspect section thickness with optical microscope

AZ9260 wafers

Material:

• 100mm silicon wafer
• AZ9260 positive photoresist - [http://www.microchemicals.com MicroChemicals GmbH]

Method:

• Oxygen plasma
• Automatized coating process using EVG150 - resist thickness: 8um
• Expose for 2x15s at 274mW using corresponding mask (inverted control mask)
• Automatized developing process using EVG150
• Hard-bake for 20min at 160°C
• Inspect section thickness with optical microscope

Masks lithography

Material:

• 5" square masks
• Chip design implemented in Clewin (.gdsi files)

Method:

• Convert Clewin files (positive resist needs to be inverted)
• Setup LASER patterning - Use 4mm head
• Develop photoresist
• Chr etch for 110s, quick rinse and ultra clean rinse
• Clean for 15min in 1165 remover hot bath and for 15min in 1165 clean bath, quick rinse and ultra clean rinse
• Air dry and inspect with optical microscope

PDMS 2 layer device fab

Materials:

• flow and control layer molds
• Sylgard 184 part A and part B
• TMCS

Method:

• Place molds into a TMCS vapor chamber
• Control layer mixture: 30g Part A + 6g Part B
• Mix for 1 minute, degas for 2 minutes
• pour onto control layer mold and place mold in vacuum chamber
• Flow layer mixture: 30g Part A + 1.5g Part B
• Mix for 1 minute and degas for 2 minutes
• Spin coat onto flow layer at 2600-3000rpm for 30secs
• Remove control layer mold from vacuum chamber, making sure no bubbles are left on the surface.
• Place the control and flow layer in a 80C convection oven and incubate for 30 minutes
• Remove casts from oven, cut out control layer, punch holes, and align to flow layer
• Put aligned device back into 80C oven and incubate for at least 90 minutes.
• Remove devices from oven and punch flow layer holes

Safety

In accordance to the guidelines of the iGEM committee, we address here the question of safety in our project. Since our project relies solely on expression in bacteria of quorum sensing molecules and fluorescent markers, it does not pose any particular threats to humans, animals or the environment, other than the ones which are encountered in standard laboratory work with E.coli. Therefore, we estimate the risk to researchers, the public or environment as very low. However, we would like to underline the particular attachment which our institution, EPFL, holds for the safety of all parties concerned by research conducted in its facilities.
As referenced on [http://biosafety.epfl.ch/page61278.html this web page], our school has Biosafety Officers, whose charges are to supervise activities linked to pathogens or genetically modified organisms. They also verify that good laboratory pratice and safety measures are respected. We have not contacted our Biosafety officer for the duration of our tasks, as they did not present any risks. If necessary iGEM teams would be able to collaborate with such safety organisations in the institution.

References

Anderson, J. C., Voigt, C. A., and Arkin, A. P., Environmental signal integration by a modular AND gate. Molecular Systems Biology 3 (2007).

Basu, S. et al., A synthetic multicellular system for programmed pattern formation. Nature 434 (7037), 1130 (2005).

Brenner, K., Karig, D. K., Weiss, R., and Arnold, F. H., Engineered bidirectional communication mediates a consensus in a microbial biofilm consortium. Proceedings of the National Academy of Sciences of the United States of America 104 (44), 17300 (2007).

Camilli, A. and Bassler, B. L., Bacterial small-molecule signaling pathways. Science 311 (5764), 1113 (2006).

Fuqua, C., Parsek, M. R., and Greenberg, E. P., Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing. Annu Rev Genet 35, 439 (2001).

Waters, C. M. and Bassler, B. L., Quorum sensing: cell-to-cell communication in bacteria. Annu Rev Cell Dev Biol 21, 319 (2005).

You, L. C., Cox, R. S., Weiss, R., and Arnold, F. H., Programmed population control by cell-cell communication and regulated killing. Nature 428 (6985), 868 (2004).

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