Team:UANL Mty-Mexico/wetlab/interlab

From 2014.igem.org

Wet-Lab

Interlab

Special Collaboration

This summer we decided to work on Interlab and tried to construct the basic parts needed. Although we tried several times to construct the device BBa_J23101 + BBa_E024, we did not achieved it. Fortunately, we keep comunication with Tec-Monterrey and found that they were having the same problem with the device BBa_J23115 + BBa_E024. Because of that the best way to finish properly our work we exchange devices.

Protocols

The protocols that we use are the same that we specify in our section “Protocols”, but in this case we describe the specific details.

Selection of DNA from distribution plates

We selected the DNA from the recommended wells to work with them.

Transformation

1. Add 50 µL of E. coli Top10 Ca+2 competent cells to a pre-chilled centrifuge tube. Keep always on ice until step 4.
2. Add 1 µL of every DNA solution to the tubes and mix.*
3. Chill the tube on ice for 20 - 30 minutes.
4. Expose the reaction mixture to a 42ºC for 1 minute heat-shock.
5. Put the tube on ice for 2 minutes.
6. Add 200 µL of antibiotic-free LB media.
7. Incubate at 37ºC for 20 - 30 minutes.
8. Spread the appropriate quantity of cells (50-200 µL) on selective LB agar plates.
9. Incubate overnight at 37°C.
10. Select colonies from the plates and culture them for 15 hours in microcentrifuge tubes with 600 µL of LB media. The tubes must have a tiny hole in the cap to allow the oxygenation.
*The parts that were transformed are BBa_I20260, BBa_J23101, BBa_J23115 and BBa_E0240.

Minipreparation

1. From the cultures, take 50 µL and store them at 4°C in new microcentrifuge tubes.
2. Centrifuge the other 550 µL at 14,000 rpm for 30 seconds. Remove the supernatant.
3. Add 200 µL of Solution I. Resuspend the pellet by using vortex briefly or by pipetting up and down. Incubate at room temperature for 5 minutes.
4. Add 200 µL of Solution II and mix gently by inverting and rotating the tube several times. Do notvortex. Incubate at roomtemperaturefor 5 minutes.
5. Add 200 µL of Solution III and mix gently by inverting and rotating the tube several times. Incubate the tube on ice for 5 minutes.
6. Centrifuge at 14,000 rpm for 5 minutes.
7. Transfer the supernatant to a fresh tube containing 1 mL of 100% ethanol.
8. Incubate at -20 °C for 10 minutes. (Max. 2 h)
9. Centrifuge at 14,000 rpm for 10 minutes. Remove the supernatant.
10. Add 200 µL of 70% ethanol and vortex gently for 10 seconds.
11. Centrifuge at 14,000 rpm for 5 minutes. Remove the supernatant by pipetting. Aspirate off any residual supernatant.
12. Dry at 37ºC for 5 minutes.
13. Add 20 µL of H2O + 20 µg/mL of RNase. Resuspend by using vortex briefly.
14. Verify the Plasmidic DNA with agarose gel (0.8%) and store at 4°C.

Digestions

We prepared the digestion reactions as described in next table.

Optionally, 5 µL of every digestion can be verified in a agarose gel 1.0 %.

Ligation

1. Prepare the ligation mix depending on the concentration of the DNA. The quantities recommended for the Software ligation calculator, of gibthon.
2. Incubate the reaction to room temperature for 1 hour or alternatively, all the night.
3. Do transformation with every ligation.

Selection of clones

1. Select random colonies of the petri dishes and culture them for 15 hours in 600 µL of LB media.
2. Save 20 µL of every tube.
3. Take 200 µL and measure the fluorescence in a plate reader. Then, the results are analyzed (see “measurements” protocol).
4. Extract the DNA of the remaining bacteria with miniprep (See “minipreparation” protocol).
5. Digest cultures with XbaI and do an electrophoresis to verify the size of the plasmids.
6. Select those clones that show fluorescence and have the correct plasmid.

Measurements

1. Cultivate the selected clones in microcentrifuge tubes during 15 hours with 600 µL of LB media. Remember that the tubes must have a hole in the cap.
2. Take 200 µL of every tube to a black 96-Wells plate (of COSTAR). Also, include a negative control (LB media).
3. Measure in a Plate reader (Biotech Synergy HT). Set the excitation filter of 460±40nm, emission filter of 528±20nm and sensibility of 60.
4. Process the data in Excel. The Optical density (OD) and fluorescence of the media is subtracted to the data. From the results, divide the highest OD to every data to obtain a correction factor. Then multiply that factor to the fluorescence to obtain the relative fluorescence.
5. Graph the resulting data.

Results

Now we present all our results obtained to Interlab.

Every set was obtained in different days.

Clones A = Part I20260 (GFP generator)
Clones I = J23115 + BBa_E0240 (Weak promoter + CDS of GFP)
Clones K = J23101 + BBa_E0240 (Strong promoter + CDS of GFP)

Means based on final results and theirs respective standard deviation.

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