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Team:Paris Saclay/Notebook/August/5
From 2014.igem.org
Contents |
Tuesday 5th August
Lab work
A - The chassis coli Odor free
PCR verification
by Romain
Results of the striates on dishes from yesterday:
For the strain MG1655Z1:
- Nothing has grown on the kan dish, but the colony grew on LB dish. That was what we expected, the kan cassette was correctly delete.
For the strain MG1655:
- Few colonies have grown on the kan dish, and all the colonies have grown on LB dish. It's a little failure.
So now, we proceed to the PCR to verify the length of the sequences.
Strains used: E. coli MG1655Z1 and E. coli MG1655, and oligonucleotides used: iPS75 bis and iPS76 bis.
Protocol
Add into a PCR tube the following:
| Component | For a total volume of 50μl |
|---|---|
| H2O | 28.25μl |
| Green GoTaq buffer 5X | 10μl |
| dNTPs 10mM | 1μl |
| iPS75 bis 10µM | 2μl |
| iPS76 bis 10µM | 2μl |
| DMSO | 1.5μl |
| MgCl2 25mM | 4μl |
| Bacterial culture | 2μl |
| Green GoTaq enzyme | 0.25μl |
We follow these quantities for 11 tubes: 5 tubes for the strain MG1655, 5 tubes for the strain MG1655Z1 and 1 tube with bacteria which had grown on the kan dish. Tube was placed in PCR machine with the following parameters.
| Cycle step | Temperature | Time | Cycle | |
|---|---|---|---|---|
|
Initial denaturation |
95°C |
10 min | ||
| Denaturation | 95°C | 30 s | 30 | |
| Annealing | 53°C | 30 s | 30 | |
| Extension | 72°C | 2 min | 30 | |
| Final extension | 72°C | 5 min | 1 | |
| Final extension | 12°C | hold | 1 |
The different bacterial cultures in each tubes:
- Tube 1 to 5: MG1655
- Tube 6 to 10: MG1655Z1
- Tube 11: MG1655 which had grown on the kan dish.
The electrophoresis results:
- 1. 10 µl of ladder
- 2 to 6. 10 µl in each tube of PCR for the strain MG1655
- 7 to 11. 10 µl in each tube of PCR for the strain MG1655Z1
- 12. 10µl of PCR for the MG1655 which had grown on the kan dish
Results of the PCR: The electrophoresis revealed correctly the expected size of the sequence, except for the holes 2 and 11. The kan resistance sequence had been successfully removed.
Liquid cultures overnight made: 3ml LB medium + 500µl transformed strain MG1655 + Cm
C - Salicylate Inducible Suppressing System
Liquid Culture
by Fabio
2 liquid cultures of BBa_K1372000 with 20ml of LB and 20µl of amp (at 3pm - 150 rpm - 37 °C), from the stock made the 4th August.
D - Lemon scent
Electrophoresis
by Sean & Pierre The strains used were made the 4th August
- 5 µl of BBa_K762100 non diluted
- 5 µl of BBa_K762100 diluted 10-1
- 5 µl of BBa_K517003
Human Pratices
by Pierre
Ethics
I wrote the begining of a global view of the evolution of the definitions of life from Aristotle to modern times.
Bibliography
- Aristotle, On the Soul
- Epicurus, Letter to Herodotus
- Descartes, Discourse on the method
- Darwin, On the origin of species
- Bichat, Physiological researches on life and death
by Hoang Vu
Continue my bibliographical research on Citral A and B and Beta-Pinene. I have now a little more than twenty reviews about various uses and properties of citral
Art & Design
by Leila & Juliette
We tested some different concentration of agar in LAG, how we can hang it, how it looks and how easy it is to turn it out : 15 g/l, 20 g/l, 25 g/l, 30 g/l, 35 g/l.
| Concentration | Strength | Color |
|---|---|---|
| 15 g/l | to much liquid : we can't hang it | really translucent |
| 20 g/l | good holding and resistance | quite translucent |
| 25 g/l | good holding and resistance | quite translucent |
| 30 g/l | good holding and resistance | not so much translucent |
| 35 g/l | really resistant. Maybe a little bit too heavy. | opaque |
Finally, we though our test were effective and we decided to select only 3 concentration for the future : 20 g/l, 25 g/l and 30 g/l.
Members there:
- Instructors and advisors: Alice and Sylvie.
- Students: Eugene, Fabio, Hoang Vu, Leila, Juliette, Pierre, Romain, Sean and Terry.
