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.

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