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Tandem OriT by Mingzhi Qu

Conjugation Test:F+_F-OriT-pSB1A2 X Dh5α_psb3K3(I)

• Donor: F+_F-OriT-pSB1A2 (Amp+)
• Recipient: Dh5α_psb3K3(Kan+)
• Control:

1. donnor：F+
2. donnor：Dh5α_OriT-pSB1A2 (Amp+)

• mixing condition: 220rpm, 90min.

• Day1:

1. Get the plates from -4 fridge：F+_F-OriT-pSB1A2 (Amp+), F+, Dh5α_OriT-pSB1A2, Dh5α_psb3K3(Kan+)#Amplification Culture in liquid LB for 12 hours.

• Day2:
• put 2mL of culture into 20mL of LB with antibiotics, sub-culturing.

Donor

• final OD600:

F+_F-OriT-pSB1A2  0.473
F+                0.456
Dh5α_OriT-pSB1A2  0.474

Recipient

• final OD600:

#Dh5α_psb3K3       1.910

Mixing

• Wash 2 times.

Plating the conjugant mix

• Only F+_F-OriT-pSB1A2 X Dh5α_psb3K3 was set up serial dilutions:original, 10-1, 10-2.
• culture on Tc+/Amp+ plate.

result(Next day)

No Donor                   X   recipient  time rpm  clones(original)  clones(e-1)   clones(e-2)
1  F+_F-OriT-pSB1A2        X  Dh5α_psb3K3  90  220     full               1000+       300+
2  F+                      X  Dh5α_psb3K3  90  220      <10                 /           /
3  Dh5α_OriT-pSB1A2        X  Dh5α_psb3K3  90  220      full                /           /