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.

Aug 08

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.

Aug 07

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.

Aug 08

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.

Aug 11

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).

Aug 12

Pseudovibrio denitrificans transformed have been still growth.

Aug 13

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.

Aug 14

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.

Aug 15

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.

Aug 16

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

Aug 17

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.

Aug 18

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.

Aug 22

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'

Aug 23

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.

Aug 24

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).

Aug 27

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.

Aug 29

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.

Sep 01

BBa_1413044 retrieval of the transposon plasmid

strategy:

Digestion of BBa_1413044 by BglII :
50 ul final volume, 5 ul Buffer NeB 3.1, 5 ul (20 ng/ul) BBa_1413044 in pSB1C3, 1 ul BglII, 39 ul H2O.
Gel electrophoresis:

Purification sur gel:
Kit thermoscientific "Gel extraction". Elution final in 30 ul.

Obtaining the Kan gene on pNK2

PCR: enzyme: Q5; template: 150 ng pNK2 first three well from new stock, next ones the old stock; oligo: F 82/ R 83; Tm tested: 55/60/65; elongation time: 1m30s

Purification sur gel:
Kit thermoscientific "Gel extraction". Elution final in 30 ul.

Oct 13

Ligation of BBa_K1413001

strategy:

Digestion of BBa_K1413001 by E/S :
50 ul final volume, 5 ul Buffer NeB 3.1, 5 ul (35 ng/ul) BBa_1413001 in pSB1C3, 1 ul E/S, 39 ul H2O. 1h
Then Thermoscientific "PCR clean up" Kit in 30 ul final elution

Digestion of Kan 82/83 by E/S & X/P:
50 ul final volume, 5 ul Buffer NeB 4.1, 5 ul (30 ng/ul) Kan 82/83 in pSB1C3, 1 ul E/S or X/P, 38ul H2O. 1h
Then Thermoscientific "PCR clean up" Kit in 30 ul final elution

Ligation:
I° strategy:
20 ul final volume, 5 ul Buffer T4 fermentas, 3 ul digestion product BBa_K1413001 by E/S + 3 ul Kan 82/83 + 5 BBa_1413044 transposon plasmid. 4 ul H2O. 1 ul T4 ligase. 1h

II° Strategy:
20 ul final volume, 5 ul Buffer T4 fermentas, 3 ul digestion product BBa_K1413001 by E/S + 5 BBa_1413044 transposon plasmid. 4 ul H2O.1 ul T4 ligase. 1h

Clean up:
II° strategy:
Ligation product by Thermoscientific "PCR clean up" Kit in 30 ul final elution

Digestion: II° strategy
50 ul final volume, 5 ul Buffer NeB 4.1, 5 ul ligation product, 1 ul E/X, 39 ul H2O. 1h
Then Thermoscientific "PCR clean up" Kit in 30 ul final elution

Ligation:
II° Strategy:
20 ul final volume, 5 ul Buffer T4 fermentas, 3 ul ligation product E/X + 5 ul Kan 82/83 E/S. 4 ul H2O. 1h

Transformation: electrocompetent

50ul electrocompetent cells 2 ul Ligation product of I° strategy / II° strategy in E.coli DH5alpha.

Oct 14

14.10.2014 ligation

it gave only one clone.

Clone culture:
5 ml LB + 5 ul Kan (25 mg/ml) - 37 C°

Transformation Pseudovibrio denitrificans & Dh5 alpha with mRFP in BBa_1413044

electrocompetent cells:

1 mm cuvette; 1800 V
recuperation media: MB for Pseudovibrio denitrificans , LB for DH5alpha

plating:

MB agar for Pseudovibrio denitrificans
LB agar for LB for DH5alpha

Transformation Pseudovibrio denitrificans & Dh5 alpha with BBa_1413001 in BBa_1413044

electrocompetent cells:

1 mm cuvette; 1800 V
recuperation media: MB for Pseudovibrio denitrificans , LB for DH5alpha


plating:

MB agar for Pseudovibrio denitrificans
LB agar for LB for DH5alpha

Verification testing of pnK2 in Pseudovibrio denitrificans

16 clones of Pseudovibrio denitrificans were grown in 3 ml MB, 30 C°, 24h

DNA extraction:

Invitrogen DNA extraction kit used with the Gram - protocol.
nanodrops gave value between 80 ng/ul & 130 ng/ul

PCR for Kan gene insertion:
PCR: enzyme: Q5; template: 200 ng genomic preparation Pseudovibrio denitrificans; oligo: F 82/ R 83; Tm tested: 55; elongation time: 2m00s

PCR for Pseudovibrio denitrificans specific oligo:
PCR: enzyme: Q5; template: 200 ng genomic preparation Pseudovibrio denitrificans; oligo: F 72/ R 73; Tm tested: 55; elongation time: 2m00s

As a control we tested the specificity of our oligo on the strain we work with:

Oct 15

Trying to show BBa_1413044 work even empty in DH5alpha

8 colonies of DH5alpha were transformed with BBa_1413044 and grown in 3 ml LB + 2 negative control

Preparation for PCR colony:
Spin down 500 ul culture at 14000 g - 2 min, resuspend in 100 ul H20 MilliQ, put at 95 C° for 10 min then spin down at 14000 g - 2 min.

PCR: enzyme: Q5; template: 3 ul of supernatent for the culture; oligo: F 100/ R 101; Tm tested: 55; elongation time: 1m00s

The two last wells are the controls which surprisingly displayed a band, and we did not obtain a single band for the 8 strains. In the end the gel revealed itself to be inconclusive with regards an empty transposon vector.

Defining where the transposase integrate

find enzyme with a good cut numbers in the genome and not in our insert: HindIII Digestion of 8 Pseudovibrio denitrificans DNA preparation by HindIII
50ul final volume, 200 ng DNA preparation, 5 ul Neb 2.1 buffer, 38 ul H2O, 1 ul HindIII. 1h

Then religate:
Ligation: 20 ul final volume, 2 ul 10x T4 ligase buffer, 1 ul T4

then light digestion with XbaI
50ul final volume, 20 ul DNA religated preparation, 5 ul Neb 2.1 buffer, 38 ul H2O, 1 ul XbaI enzyme. 15 min
deactivation at 80 C° for 20 min

then PCR
PCR: enzyme: Q5; template: 3 ul of digestion by XbaI; oligo: F 105/ R 106; Tm tested: 55; elongation time: 4m00s

One clone clearly displayed numerous insertions, while the others having fewer bands tend to show band sof the same size, which could prefigure the existence of favourite spot, as can be seen in E.coli.

With further experiments and sequencing run we will be able to precisely locate the insertions and the better define how many times an insertion event can occur at a given concentration of plasmid electroporated.

Oct 16