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.
Verication of Cell Surface Display
1. SDS-PAGE
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.
Sample Preparation
1.Centrifuge 1ml culture medium/bacteria liquid at 12000rmp for 2 min to separate the supernatant and cells.
2.Add 100μl DNase/RNase-Free Water to the precipitation, then add in 100μl 2×loading buffer.
3.Place tubes in 100℃ heat block for 10 min.
4.Centrifuge at 12000rmp for 2 min.
Separating Gel:
| Components |
Volume |
| 1.5M TrisHCl pH 8.8 |
3.8 mL |
| 30% Acrylamide 0.8% Methylene bis Acrylamide |
6.0 mL |
| ddH2O |
4.9 mL |
| 10% SDS |
150 uL |
| 10% (NH4)2S2O8 |
150 uL |
| TEMED |
6 uL |
Separating Gel:
| Components |
Volume |
| 1.5M TrisHCl pH 8.8 |
750 uL |
| 30% Acrylamide 0.8% Methylene bis Acrylamide |
1.0 mL |
| ddH2O |
4.1 mL |
| 10% SDS |
60 uL |
| 10% (NH4)2S2O8 |
60 uL |
| TEMED |
6 uL |
1. Make the separating gel:
Set the casting frames (clamp two glass plates in the casting frames) on the casting stands. Prepare the gel solution in a separate small beaker.Swirl the solution gently but thoroughly. Pipet appropriate amount of separating gel solution into the gap between the glass plates.To make the top of the separating gel be horizontal, fill in water into the gap until a overflow.Wait for 20-30min to let it gelate.
2.Make the stacking gel:
Discard the water and you can see separating gel left. Pipet in stacking gel untill a overflow. Insert the well-forming comb without trapping air under the teeth. Wait for 20-30min to let it gelate.
3. Make sure a complete gelation of the stacking gel and take out the comb. Take the glass plates out of the casting frame and set them in the cell buffer dam. Pour the running buffer (electrophoresis buffer) into the inner chamber and keep pouring after overflow untill the buffer surface reaches the required level in the outer chamber.
4. Load prepared samples into wells and make sure not to overflow. Don't forget loading protein marker into the first lane. Then cover the top and connect the anodes.
5. Set an appropriate volt and run the electrophoresis when everything's done.
6. As for the total running time, stop SDS-PAGE running when the downmost sign of the protein marker (if no visible sign, inquire the manufacturer) almost reaches the foot line of the glass plate. Generally, about 2.5 hours for a 100V voltage and a 12% separating gel.
7. Stain the gel with Coomassiae billiant blue R250 for 4 hours and destain the gel with water at least 2 hours.
2. Fluorescence immunoassay to verify the cell surface display
(1) The fermentation of recombinant E.coli.
(2) Aspirate 1 mL culture to an centrifuge tube with 20 mL LB. Inoculate the culture at 37℃ with shaking (rotate speed:200rpm) till the OD at λ=600 is between 0.4 and 0.6.
(3) Add 20 uL IPTG at 0.1 mol/L into the culture. Inoculate the culture at 28°C with shaking (rotate speed:200rpm) for 10 hours.
(4) Aspirate 1 mL cell suspension to centrifuge tube. Pellet the suspension by centrifugation at 4000 rpm, 4℃,2 min. Discard the supernatant.
(5) Use 1 mL PBS buffer (pH=7.4) to resuspend the cells. Pellet the suspension by centrifugation at 4000 rpm, 4℃ for 2 min. Discard the supernatant. Repeat this step twice.
(6) Resuspend the cells with 1 mL 5% BSA solution, add 1uL DYKDDDDK Tag (9A3) Mouse mAb (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody). Seal the centrifuge tube the parafilm.
(7) Blend the cell suspension. Put the tube in the ice overnight standing for 4℃.
(8) Pellet the suspension by centrifugation at 4000rpm, 4℃ for 2 min. Discard the supernatant.
(9) Use 1mL PBS buffer (pH=7.4) to resuspend the cells. Pellet the suspension by centrifugation at 4000rpm, 4℃ for 2 min. Discard the supernatant. Repeat this step twice.
(10) Resuspend the cells with 1mL PBS buffer，add 1μL Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) .
(11) Blend the suspension. Seal the centrifuge tube the parafilm and wrap it with tinfoil. Put the tube in the ice and incubate the cells for 1~2h (blend the suspension with inverting at times during the period).
(12) Pellet the suspension by centrifugation at 4000rpm, 4℃ for 2 min. Discard the supernatant. Repeat this step twice.
(13) Suspend the cells with 300μL PBS. Cover it with coverslip. Use the fluorescence microscopy to observe under the excitation light of 488nm.
The preparation of PBS pH7.4:
NaCl 8.00g/L
KCl 8.00 g/L
Na2HPO4 3.58g/L
KH2PO4 0.244g/L
Use sterile filter membrane (pore size=0.22 um) to filtrate bacteria.
Verification of Tb3+ capturing by precipitation titration of remained Tb3+
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.
