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Week 7: 29th July - 4th August

Construction of plasmid N°1

We make an electrophoresis with 5 µL of plamsid to check the plasmid purification made on the last friday.

Mettre l'image légender ici

There is not the 3 bandes that we sould see, so to check another time, we make a digestion with Pst I and EcoR I;

Distilled water 50 µl

NEB Buffer 10X 5 µl

BSA 10X 5 µL

DNA 2 µg

Pst I 2 µL

EcoR I 2 µL

After another electrophoresis, we obtain no band.

Fuck

Construction of plasmid N°2

Our plasmid N°2 is building with a synthetic promote sequence which is composed of:

• Andersen's promotor
• Fur Binding Site (15 different)
• RBS
• sfGFP
• Terminator

Golden Gate

In order to associate these sequences we performed a golden gate, using for each sample :

• 80 ng of Andersen's promotor
• 80 ng of Fur Binding Site
• 80 ng of RBS
• 80 ng of sfGFP
• 80 ng of Terminator
• 1.5 µL of T4 buffer (10X)
• 15 Unit of T4 ligase
• 2.5 Unit of Bsa I

The Golden Gate products are chemically transformed into E. coli Top 10 strains. After the transformation process, bacteria are plated into LB medium with carbenicillin and icubated overnigth at 37°C.

Construction of plasmid N°3

29/07/13
We received the primers ordered on friday the 26th of July and started to dilute them into TrisCL at 10 mM. Secondly, we diluted the previous stock solution at a rate of 1/20th to obtain a final concentration of 5 µM for our intermediate solution.

Then, for our plasmid three construction, we need to first extract the 6 enterobactin gene (EntA, EntB, EntC, EntD, EntE and EntF). The ordered primers from friday will theoretically extract them in the appropriate Golden Gate format with their own RBS upstream (designed from Salis RBS). This will allow us to construct our two N°3 plasmids, one containing EntA, EntD and EntF, and the other one EntB, EntC and EntE. The genes are spread like this to obtain two equivalent plasmids xxx

We proceeded to a genomic extraction of the 6 genes of interest and migrated to PCR products on a 1% gel. We successfully extracted 5 out of the 6.

30/07/13
We annealled the oligonucleotides of the PL-LacO part in the golden gate format. Also, we extracted the sfGFP with the RBS upstream which will allow us to construct the control positive plasmid 3 for future TECAN experiments.