Team:UNITN-Trento/Protocols

1. Sample Capture:
   • Add 250 µl of Capture buffer type 3 to the sample.
   • Mix and control that the color is yellow or pale orange.
2. Sample Binding:
   • Transfer the mix in the assembled GFX MicroSpin column and Collection tube.
   • Spin for 30s at 16000*g (⁓13000rpm) and discard the Collection tube.
   • Replace GFX MicroSpin column in the same Collection tube.
3. Wash and Dry:
   • Add 500 µl of Wash buffer type 1.
   • Spin for 30s at 16000*g (⁓13000rpm) and discard the Collection tube.
   • Transfer the GFX MicroSpin column in a clean tube.
4. Eluition:
   • Add 50 µl of distilled water.
   • Incubate for 1 minute at room temperature.
   • Spin for 1 minute at 13000rpm.
   • Retain flowthrough.
   • Quantify the sample at Nanodrop and label it with its concentration.

• Transformation Buffer: sterile 10 mM Tris-HCl, pH 7.0, 50 mM CaCl2
• Grow a 50 mL culture in LB at 37 deg C from 1 colony.
• When OD ~ 0.5, collect the cells in a sterile Falcon tube and chill on ice for 10min.
• Centrifuge at 5000 rpm for 10 min at 4 deg C. Discard supernatant.
• Resuspend cells in 15 mL of transformation buffer.
• Chill on ice for 15 min. Spin at 5000 rpm for 10 min at 4 deg C. Discard supernatant.
• Resuspend cells in 4 mL of transformation buffer.

• Thaw home made CaCl2 competent cells on ice.
• Add 1µL of DNA to 200µL of competent cells (concentration of DNA stock should be between 50–150 ng/µL).
• Incubate on ice for 30 min.
• Heat shock at 42°C for exactly 2 min.
• Incubate on ice 1 min.
• Add 500-700µL of LB (or SOC) and shake at 37°C for 1 h.
• Plate the cells (use plates with the appropriate antibiotic according to your plasmid).

• Incubate the plates O/N upside down at 37°C.

1. Pick up a nice big colony and drop it in 2 ml of completed MC (1x) (see below);
2. Grow at 37 °C for 5 hours (or more if culture is not really turbid);
3. Mix 400 ul of culture with DNA (usually 1 ug) in fresh tube (i.e. 15 ml tubes losely closed);
4. Grow for additional 2 h at 37 °C;
5. Plate all on selective antibiotic plates, and incubate at 37 °C O/N

1. Transfer 100 ul of sterile distilled water in an eppendorf;
2. Pick up a colony using a tip and resuspend it in the sterile water;
3. Verify that the glass slide is cleaned and degreased or clean it with 70% alcohol;
4. Transfer 20 ul of bacterial suspension on the slide;
5. Swipe gently bacterial suspension with the aid of a sterile loop to occupy 1-2 cm at the center of the slide;
6. Let dry the slide by evaporation;
7. Cover the central part of the slide with methanol, remove the excess and let it evaporate;
8. Sock the slide in the crystal violet solution for 1 min, wash then with sterile water;
9. Sock the slide in the lugol solution for 1 min, wash then with sterile water;
10. Pour Gram bleach solution on the product for 20-30 s, wash then with sterile water;
11. Sock the slide in the safranin solution for 1 min, wash then with sterile water;
12. Finally let dry the slide and observe it with the microscope.

1. Label the empty eppendorfs that will contain the parts, including antibiotic resistance, part denomination and position (and on which kit).
2. Spot the correct well and label it with a pen.
3. Push a hole with the pin of a micropipette and resuspend the content with 10ul water.
4. When the color is dark red, wait 1 minute.
5. Move the re-suspended part into the correct empty eppendorf.

Prepare the digestion mix as follow:

Incubate the reaction mix at 37 °C for 30 min. Disactivate then the enzymes incubating the mix at 80 °C for 20 min. The next step will be the ligation of the digestion products. The raction mix is prepared as follow:

Gently mix the reaction and incubate for 30 min at room temperature. Disactivate the enzymes at 80 °C for 20 min. Transorm 10 ul of the reaction in competent cells.

Classic Cloning - for PCR products

Classic Cloning - for plasmids

Biobricks Cloning

Screening

An overnight culture was diluited 1:100 and grown until O.D.600 reached 0.5. After that, the culture was induced with 5 mM arabinose and placed with a stirrer in a sealed vial (V = 15 ml) with a pierceable septum. The sample was kept for about 4 hours at 37 °C in thermoshaker. After that the sample was connected to the Micro Gas Chromatograph Agilent 3000A endowed with two colums: a Mol Sieve 5A Plot and a Plot U column (see the tables for colums and method specifications).

An overnight culture was diluited 1:100 and grown until O.D.600 reached 0.5. After that, 3 ml of culture induced with 5 mM arabinose was placed with a stirrer in a sealed vial (V = 15 ml) with a pierceable septum. The sample was then connected to the Micro Gas Chromatograph Agilent 3000A endowed with two colums: a Mol Sieve 5A Plot and a Plot U column (see the tables for colums and method specifications).

To detect if E. coli actually produced Methyl salicilate (MeSA), we tried both qualitative (SNIFF Test) and quantitative (GC-MS) tests.

All the measurement have to start by inocula in the correct antibiotics (CM) of MeSA producing cells and of normal Neb10β in LB without antibiotic as control. The inocula need to be growth O/N at 37°C in agitation. The following day, dilutions 1:100 were done in falcons with fresh LB and the same antibiotic; the cells were growth at 37°C in agitation until O.D.600≈0.6 was reached.

Then the cells were induced by adding Arabinose 5mM by a solution 1 M prepared by dissolving 0,150 g in 1 mL of dH2O. After two hours in some of the samples was added Salicylic Acid 2mM by a solution 1 M obtained by dissolving 0,138 g in 1 mL ethanol 70%. Then the culture were put for other 2 hours at 37°C in agitation waiting for the 4 hours of induction to pass.

The tests could also been in M9 medium because it smells less than LB. To do this, when cells in LB reached an O.D. of 400 they were centrifuged at 4100 rpm for 10 minutes to form a pellet. The supernatant was discarded and they were resuspended in fresh M9 medium with the correct antibiotic. This was done because E.coli cells growth in M9 in much more time than in LB. The cells were therefore induced with Arabinose and after two hours SA was added where needed.

To have a quantitative analysis we used the Finnigan Trace GC ULTRA with a flame ionization detector (FID). To achieve the final results and finally measure the quantity presents in our samples many tries were done with different conditions and methods. For what concerns the results presented in our wiki the column exploited was a DB5-MS capillary with a total length of 30 m (loops), an internal diameter of 0.25 mm and a film of 0.25 μm.

The temperature program set for all the analyses was taken from literature (Deng, C, et al. Investigation of Tomato Plant Defence Response to Tobacco Mosaic Virus by Determination of Methyl Salicylate with SPME-Capillary GC-MS. Chromatographia. 2004, Vol. 59, 3/4, pp. 263-268) and adapted to our experiment in order to decrease the time of each measurement. In particular the program was set to start with the column at 80°C for 2’, to increase the temperature at the rate of 30°C/min until reaching the temperature of 280°C and maintaining it for 10’. The temperatures of the injector and of the FID were set 280°C. The carrier in the column was He 1.4 mL/min and the flow of the FID was set to be 40 mL/min of H2, 450 mL/min of air and to make up 30 mL/min of N2. The modality of the introduction of the samples in the detector was set to splitless, in particular 70 mL/min. With these setting the retention index of MeSA was between 10.20-10.27 min.

The measuraments were done on liquid (the tries done on the headspace of a standard sample by injection of 1 ul of air were not positive). In particular each bacteria sample was before centrifuged and 1 mL of supernatant was filtrated with a 0.22 μm filter and then 1 μL of each sample was injected in gas chromatography to not damage the instrument. Before and after each injection the syringe was washed with acetone or ethanol for 3 times to eliminate any residual.