Cloning methods
A.The PCR Reaction System
| Components (50μL) |
Volume(μL) |
| 5×phusion HF Buffer |
10 |
| dNTPs(2.5mM) |
5 |
| Primer-F(10μM) |
2.5 |
| Primer-R(10μM) |
2.5 |
| DMSO |
1.5 |
| phusion |
0.5 |
| ddH2O |
27.5 |
| Template |
0.5 |
B.The double enzyme digestion system (Q.cut)
| Components (30μl) |
Volume(μl) |
| 10 x Q.cut buffer |
3 |
| Substrate |
10 |
| Enzyme I |
1 |
| Enzyme II |
1 |
| ddH2O |
15 |
| Conditions |
37℃ 1.5~2h |
C.Ligation system
| Components (10μl) |
Volume(μl) |
| T4 ligase |
1 |
| T4 ligase buffer |
1 |
| Linearized Vector |
1 |
| Insert Gene |
7 |
| Conditions |
16°C 1h |
D.The SOE（Gene splicing by overlap extension） PCR
We use SOE to ligate different genes, which are difficult to cut or ligate. We achieve it by overlap extension during PCR.
First, we need to design 4 primers. Primer 1 & 4 are normal ones. The left part of primer 2 is normal primer, while the right part is primer for gene B. Primer 3 is just like primer 2. The right part of primer 3 is normal, while the left part is primer for gene A. Some base of primer 2 and primer 3 can reach complementary base pairing.
Second, we amplify gene A by primer 1 & 2 and amplify gene B by primer 3 & 4 respectively. After that we can get extended gene A and gene B.
Last but not least, we blend extended gene A & gene B can complemet each other and form the hybirdized strands. Under the function of DNA polymerase I, gene A and gene B can be the primer and templet of each other. After PCR, we can get fused Gene A-B.
E.Transformation into E. coli DH5α
The ligation product was transformed into E.coli DH5α strain.
If the vector was pET28a, the strain was grown in LB plate medium containing 10μg/ml kanamycin at 37°C.
If the vector was pBAD30, the strain was grown in LB plate medium containing 100μg/ml ampicillin at 37°C.
If the vector was pSB1C3, the strain was grown in LB plate medium containing 25μg/ml chloramphenicol at 37°C.
Construction of the whole circuit
The protocol of SDS-PAGE:
1. Take 1ml of the bacterial suspension(OD600 is about 2.0), add 9ml LB medium, culture the bacteria in a shaker until its OD600 is 0.6.
2. Add 1 ‰ IPTG to induce our engineering bacteria, and culture them for 5 hours.
3. Centrifuge the bacterial suspension, and gently rinse it, add utrapure water into the cultures until its OD600 is about 2.0.
4. Prepare different concentrations of terbium chloride solution of 40ml. Adding 3ml bacteria suspension to each of them respectively.
5. Add excess sodium hydroxide to the supernatant until all the ions are precipitated. Centrifugate.
6. The resulting precipitate was titrated with hydrogen chloride to give the titration results.
Titration
We transferred the plasmids carrying the sequences encoding 3×LBT into BL21, and set a control group in which 3×LBT is not involved in the plasmids transferred. Both can be expressed with the induction of IPTG. Having cultivated the bacteria into similar densities, we add IPTG and then incubate them for 5 hours. Afterwards, we boiled the bacteria to release the proteins on their cell membrane. Then we detected the proteins we extracted by SDS-PAGE. As a result, bacteria in the test group expressed a large quantity of LBT while those in the control group did not.
