Notebook

Protocols

Recombinase overexpression and extraction

Pre-culture overnight of the strain containing recombinase plasmid at 37°C.
Culture at 37°C for 4 hours.
Centrifuge the culture for 3 min at 13000 rpm.
The cell pellets were resuspended in 600µl Rec buffer 1 (for Bxb1 and FimE recombinases) or Rec buffer 2 (for Tp901 and PhiC31 recombinases).
Sonication of the cells for three bursts of 30s.
Centrifugation for 15 min at 13000 rpm, 4°C, the supernatant containing proteins (recombinases) was collected and incubated on ice.

Rec buffer 1 : 20 mM Tris, pH 7.5, 10mM EDTA, 25mM NaCl, 10mM Spermidine, 1mM DTT and 0.1g.mol-1 BSA.
Rec buffer 2 : 20 mM Tris, pH 7.5, 1mM EDTA, 1M NaCl.
Notes : For overexpression of Tp901 and Bxb1 from Dual Controller plasmid of Bonnet, 20 ng/ml of aTc and 1% of arabinose were added to the culture in order to activate the pTetO and the pBAD promoters.

In vitro recombination test Bxb1¹ recombinase

Recombination reactions were assembled on ice with 100 ml² of Bxb1 recombinase extraction solution and 20 ml of solution containing Rec buffer 1³ and 60 ng⁴ the gate plasmid.
Reactions were incubated at 37°C⁵.
Taking sample (20 µl) for different time from 15 min to 5 hours⁶ and heat inactivated at 75°C for 10min.
Reaction taking samples were transformed into E.coli K12-DH5.1.
Spread out the cells on LB agar plate containing the antibiotic to which the gate plasmid contains the resistance gene.
The plate was incubated overnight at 37°C⁵.
In order to RFP expressed can detected by human eye, the plate was exposed after in room temperature for 16 hours up to 32 hours.

The same protocol is used with different conditions depending on the recombinase(s) used:

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