Molecular cloning: Pcat-2M-lacI/tetR-term-lacP/tetP+GFP
Parts: K228817/18+E0840=K228819/20
Resource
Pcat-2M-lacI/tetR-term-lacP/tetP (K228817/18): myself, colonies, renamed as L1, L2, L3, T1, T2 and T3.
GFP (E0840): from Lin Min, vector (has already digested by EcoR1 & Xba1)
2009.8.5
Plasmid mini prep:
L1, L2, L3
Double digest:
L1, L2 and L3:
| EcoR1 | 1uL
|
| Spe1 | 1uL
|
| plasmid | 10uL
|
| Buffer | 2uL
|
| water | 6uL
|
37 ℃ 4 hour
Gel electrophoresis:
Products of double digest of L1, L2 and L3,
marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
loading buffer and DNA dye: 6×
voltage and time: 60V 5min; 120V 60min
lane1: Marker
lane2~4: L1~3;
The insert is about 1.4kb.
DNA Gel purification:
L1, L2 and L3
2009.8.6
DNA ligation:
| System | 10uL
|
| Insert | 6uL
|
| vector | 2uL
|
| water | 3uL
|
| buffer | 1uL
|
| T4 DNA ligase | 1uL
|
16℃ 4 hour
Insert: L1, L2;
Vertor: GFP
Transformation:
Products of ligation, competent cells 50uL each,
Smear to LB plate with Amp
Double digest:
T1, T2 and T3:
| EcoR1 | 1uL
|
| Spe1 | 1uL
|
| plasmid | 10uL
|
| Buffer | 2uL
|
| water | 6uL
|
37 ℃ 4 hour
Gel electrophoresis:
Products of double digest of T1, T2 and T3,
marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
loading buffer and DNA dye: 6×
voltage and time: 60V 5min; 120V 15min
Lane 1, 2, 4: T1~3
Lane 3: Marker
DNA Gel purification:
T1, T2 and T3
2009.8.7
Every plate (L1,L2 +GFP) is very well: more than 100 clones
And many colonies are become green under the blue light, which means that the expression of LacI can not fully repressed the promoter lacP.
The second picture is for comparison with no GFP colonies.
PCR: (colony PCR)
| Master mix | 5ul
|
| primer (standard primer) | 0.5uL each
|
| water | 4uL
|
| template |
|
6 colonies of L+GFP
Gel electrophoresis:
Products of PCR
marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
loading buffer and DNA dye: 6×
voltage and time: 60V 5min; 120V 15min
Lane 1: Marker;
Lane2~7: L1~6;
The correct insert is about 2.4kb, and we found that L1, L2, L3, L4, and L6 are all correct.
DNA ligation:
| System | 10uL
|
| Insert | 6uL
|
| vector | 2uL
|
| water | 3uL
|
| buffer | 1uL
|
| T4 DNA ligase | 1uL
|
16℃ 4 hour
Insert: T1, T2;
Vertor: GFP
2009.8.8
Transformation:
Products of ligation (T1+GFP, T2+GFP), competent cells 50uL each,
Smear to LB plate with Amp
2009.8.9
Every plate (T1,T2 +GFP) is very well: more than 100 clones
And many colonies are become green under the blue light, which means that the expression of tetR can not fully repressed the promoter tetP.
The second picture is for comparison with no GFP colonies.
2009.8.10
Plasmid mini prep:
T1+GFP, T2+GFP, T3+GFP;
Digest: (T1-G, T2-G)
Double:
| EcoR1 | 1uL
|
| Pst1 | 1uL
|
| plasmid | 4uL
|
| Buffer | 2uL
|
| water | 12uL
|
Single:
| EcoR1 | 1uL
|
| plasmid | 4uL
|
| Buffer | 2uL
|
| water | 12uL
|
37 ℃ 4 hour
Gel electrophoresis: (to confirm the T1-G, T2-G)
Products of digest
marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
loading buffer and DNA dye: 6×
voltage and time: 60V 5min; 120V 15min
Lane 1 & 5: plasmid, T1-G, T2-G
Lane 2 & 6: single digest T1-G, T2-G
Lane 3 & 7: double digest T1-G, T2-G
Lane 4: Marker
The insert is about 1.8kb, and the vector is about 2.1kb. It is very hard to separate them, yet from the gel, we know that the T1-G and T2-G are correct.
Result & Discussion
I successfully constructed the two clones: Pcat-2M-lacI/tetR-term-lacP/tetP-GFP (K228819/20).
However, I disappointed to find that these clones are not work very well, because the GFP express significantly even on the plate (without induce)!!! That means the expression of lacI and tetR are not enough to repress the lacP and tetP. It is possible that the LVA tail of lacI and tetR make they degrade very soon. (for more information of LVA refer to parts C0012 and C0040). And other possibility is that the constitutive promoter Pcat is not strong enough.
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