It’s the first day where Brian Jester gave us the plasmid pNK2. This plasmid contains transposon.
A Precultur of DH5α pir is realized.In a 15 mL tube, add 2 mL of LB medium and inoculate cells from glycerol. These cells can replicate the plasmid because they contains pi protein.
It parallel annotation of plasmid pNK2 with Blast is realized.

The electrocompetent DH5α pir is maked in 400mL of LB.
The transformation of this cells with plasmid pNK2 is effected and the cells is sowed in LB kanamycine 25µg/mL.

Colonies in the plate DH5α pir with pNK2 is put in culture in 3mL of LB add kanamycin (25µg/mL) and the plasmid pNK2 is purified.

A stock of the cells DH5α pir with pNK2 is performed.
The plasmid pNK2 is purified from precultur of DH5α pir transformed. A kit of Macherey-Nagel of Plasmid DNA purification is used.
DH5α pir transformed is sowed in LB kanamycin and one glycerol is making.

Transformation of Pseudovibrio denitrificans

Transformation of Pseudovibrio denitrificans (PseudoD) with pNK2 is realised, with Electric shock of 2000V .This voltage had been optimised in the part transformation. As Pseudovibrio lives with kanamycine at 25µg/mL, the cells is sowed in Marine Broth (MB) 1X with kanamycin 50µg/mL.
It parallel BL21 is so transformed with same plasmid but with electric shock of 1800V and sowed in LB 1X add kanamycin (25µg/mL).

Pseudovibrio denitrificans transformed have been still growth.

Verification of transformed cells

There are some colonies in the plate PseudoD transformed.
PCR 16S of PseudoD cell transformed with pNK2 is actualized. We used primer 1 and 2.
This PCR is purified by means of Macherey-Nagel Kit and send to sequence.

To confirm that PseudoD is transformed, a PCR is realized with primer 112R 112F and Q5 high fidelity.
This development did not work. There is no band revealed on the gel.

Verification of insertion pNK2 in PseudoD with other primers 65F and 65R.
This development did not work. There is no band revealed on the gel.

The map of the first plasmid pNK2 isn’t ok. There was mistake because this isn’t the RFP in the transposon but emGFP.
To verified the insertion we used a new couple of primer 42b and 92c.
This approach did not work. There is no band revealed on the gel. So this new primers which amplified the kanamycin in the transposas is bought.

A preculture of DH5α pir with pNK2 and PseudoD is performed.

Measure of GFP

A measure of fluorescence (GFP) is realised by means of TECAN. This Tecan isn't concluding.

Verification of Pseudovibrio transformed

A result of sequencing of PseudoD with transposon is received. The sequence of 16S pseudoD is compared with the sequencing. There are 100% of identity. So it's PseudoD , which is transformed.

Induction of mGFP

The promoter front emGFP is inductible by glycerol (8%). So a test of induction has been realized. Different cells is cultivated with LB 1X and glycerol 8% add kanamycin (25µg/mL) for DH5α pir and Bl21 transformed and MB 1X and glycerol 8% add kanamycin (50µg/mL) for PseudoD.

Universal Plasmid

We decided to create a new Universal Transposon Plasmid. For this we designed some primers to amplified OriVR6Kgamma, the transposas Tn10 in the format biobrick, to amplified OriVR6Kgamma transposas and the 2 Is10 to build the Universal plasmid with Golden Gate method.

First it is necessary to muted the site XbaI in the Orivr6kgamma. For this the method of PCR mutagenes is used. For this the primer is designed to amplified all the plasmid.
The site Xba I : 5' TCTAGA 3' is modified by 5' ACTAGA 3'

PCR of cell Pseudovibrio with transposas, with primers: 65F / 65R (which correspond to cassette of resistance kanamycin). 42b / 92c 112F / 112R,
is begun again, but the Tm is decrease 52°C instead of 55°C. It don't work.
The PCR didn't work.

PCR mutageneses of OriVR6Kgamma

The PCR mutagenes is realized with primers 70 and 71. The template is pNK2 purified, with standard protocol of amplification except that the Tm is 60°C and the time of elongation is of most longer (size of plasmid = 6,4 kb).

OriVR6Kgamma mutation

The PCR product is treated with Dpn1 during 2h. This enzyme cuts methylated DNA.
The transformation of DH5α pir is realized. But the cells arc all the time. The problem provides to PCR product.

Purification of pNK2 with Ori muted

The product of PCR is purified with dialyse during 2h.

Competent cells

To do sorbitol 0,20g/L and glycerol 10%.
Pseudovibrio competent with wash sorbitol and glycerol in M9 medium.