IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-8

OriT by Qu Mingzhi

Conjugation Test:R751_pSC101 X Dh5α_psb1A2

• Donor: C600-R751_psc101 (Tc+)
• Recipient: Dh5α+ psb1A2 (Amp+)
• Control:

1. donnor：C600_R751
2. donnor：Dh5α_pSC101

• mixing condition: 220rpm vs. 60rpm; 60min vs. 120min

• Day1:

1. Get the plates from -4 fridge：C600-R751_psc101(Tc+), Dh5α_psb1A2(Amp+), C600_R751, Dh5α_psc101
2. Amplification Culture in liquid LB for 12 hours.

• day 2:

1. put 2mL of culture into 20mL of LB with antibiotics, sub-culturing.

Donor

• According to the conjugation protocol
• final OD:

1. R751+psc101 1.750
2. R751 2.274
3. Dh5α+psc101 1.883

Recipient

1. According to the conjugation protocol

• final OD:

1. Dh5α+pSB1A2 2.066

mix condition

No   Donor                          rpm        time(min)
1    R751+psc101_stationary         220        120
2    R751+psc101_stationary         220        60
3    R751+psc101_stationary         60         120
4    R751+psc101_log                220        120
5    R751                           220        120
6    Dh5α+psc101                    220        120

Plating the conjugant mix

• All mix were 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  R751+psc101_stationary  X  Dh5α_pSB1A2  120  220     2000+              300+        <5
2  R751+psc101_stationary  X  Dh5α_pSB1A2   60  220     2000+              200+        <5
3  R751+psc101_stationary  X  Dh5α_pSB1A2  120  100     2000+              500+        <20      
4  R751+psc101_log         X  Dh5α_pSB1A2               2000+              <100        <10
5  R751                    X  Dh5α_pSB1A2  120  220     50+                 <10        <3
6  Dh5α+psc101             X  Dh5α_pSB1A2  120  220    2000+               200+        0   

• test failed because control received clones....they thould not grown on Tc+/Amp+ plate!!

Amplification Culture of OriT_J23066_OriT_pSB1A2(normal T4 ligase)

• select Positive OriT_J23066_OriT_pSB1A2 Colonies from Plate, Culture in liquid LB,waiting for mini-prep.

Lock & Key by Yu Tao

PCR R0010<-J23066

• vr and vf2 primer (standard sequencing primer): stored as 100uM.
• PCR system contains:

0.5 µL    Template
1 µL      Primer pf1
1 µL      Primer pr1
4 µL      dNTP
0.5 µL    Taq
5µL       10 X buffer
38µL      ddH20
--------------------------
50 µl      Total

• Primer final concentration 1uM.
• PCR program setting:

Step1     94℃ 5min
Step2     94℃ 30s 
Step3     60℃/62℃ 30s
Step4     72℃ 90s 
Step5     Go to step 2 for 29 times 
Step6     72℃ 10min
End

Electrophorsis Result

• from left to right:

2. marker

Key1 & Lock1 Efficiency Test

• Preculture the following 4 groups of DH5a overnight.

1. I7100-DH5a
2. R0010.J23066(Key)-DH5a
3. R0010.J23066(Key)+R0040.J23078.E0040.B0015(Lock) -DH5a
4. DH5a negative control.