Team:Tianjin/GFP
1.Get the gene of BFP
1.1 In order to get the fusion protein, we use overlap PCR to construct the fragment(BFP- sJanus & BFP-sJanus-m).We get the gene of BFP by the standard plasmid from iGEM. Then we use PCR to amplify it. The primer we design is as follow:
F:5’- GAATTCGCGGCCGCTTCTAGATGAGCGAACTGATCAAAGAG-3’
(EcoRI and XbaI site are contained).
R:5’- GTGGTGGTGGTCTTGGTGGTGGTGGATTCAGTTTATGACCCAGCTT-3’
(linker-GGGGSGGGGS is contained)
We get the target sequence by PCR at the temperature of 55 ,60 and 65 centigrade.
1.2 detail of the experiment
PCR system：（50μl）
|
Buffer |
5μl |
|
dNTPS |
5μl |
|
BFP_F primer |
2μl |
|
BFP_R primer |
2μl |
|
BFP PSB1C3 |
1μl |
|
Pfu enzyme |
0.5μl |
|
ddH2O |
34.5μl |
|
95℃ |
5min |
|
95℃ |
30s |
|
1：55℃ 2：60℃ 3：65℃ |
30s |
|
72℃ |
2min |
|
72℃ |
10min |
|
4℃ |
Forever |
2.Get the gene of BFP- sJanus & BFP-sJanus-m
2.1We get the gene of BFP- sJanus & BFP-sJanus-m by the plasmid pET28a(+) extracting from DH5α. And then we amplify it by PCR. The primer we design is as follow:
F:5’-ACCACCAAGACCACCACCACCAAGAAGCAACGGCAACGGCAAT-3’
(linker-GGGGSGGGGS is contained)
R:5’-CTGCAGGCGGCCGCTACTAGTATCAAGCACCGACGGCGG-3’
( SpeI, NotI, and PstI site are contained)
We get the target sequence by PCR at the temperature of 55, 60 and 65 centigrade.
2.2 Details of experiment
PCR system：（50μl）
|
Buffer |
5μl |
|
dNTPS |
5μl |
|
P_F |
2μl |
|
P_R |
2μl |
|
sJanus PET28a |
2μl |
|
Pfu enzyme |
0.5μl |
|
ddH2O |
33.5μl |
|
95℃ |
5min |
|
95℃ |
30s |
|
1：55℃ 2：60℃ 3：65℃ |
30s |
|
72℃ |
2min |
|
72℃ |
10min |
|
4℃ |
Forever |
2.3 Results
Figure 1 The maximum of marker is 2000bp, and the centigrade of 60 is not suitable for the system because there is a track behind it. Our target is about 250bp approximately
We get the target fragment in the end. All temperature were suitable for the PCR cycle.
3.Overlap PCR
3.1 We use the technique of overlap PCR to get our target sequence. In this experiment, we add BFP and sJanus & sJanus-m which we get in two steps and the BFP’s primer we design and sJanus & sJanus-m’s reverse in the system of PCR. We get the target sequence at the temperature of 55, 60 and 65 centigrade.
3.2 Detail of the experiment
PCR system（100μl）
|
Buffer |
10μl |
|
dNTPS |
10μl |
|
BFP fragment |
5μl |
|
sJanus fragment |
5μl |
|
Pfu enzyme |
1μl |
|
ddH2O |
69μl |
Add two kinds of primer (1μl) after 5 cycle.
|
95℃ |
5min |
|
95℃ |
30s |
|
1：50℃ 2：56℃ 3：65℃ |
30s |
|
72℃ |
2min |
|
72℃ |
10min |
|
95℃ |
5min |
|
95℃ |
30s |
|
1：50℃ 2：56℃ 3：65℃ |
30s |
|
72℃ |
2min |
|
72℃ |
10min |
|
4℃ |
Forever |
3.3 Results
Figure 2
Figure 3. The maximum of marker is 2000bp, and following is 1000, 750, 500, 250 and 100bp from up and down, our target sequence is about 1000bp approximately. We failed at first attempt. But as we change the temperature we use in PCR cycle. We finally get the target fragment.
As we can see from the picture, there are two bands in every channel. One is about 1000bp and another is about 300bp. We do not know the reason of this. But after doing the confirmatory experiment, we are sure the 1000bp band is the target band we want (BFP- sJanus & BFP-sJanus M). We did not get the fragment at first attempt. We change the temperature in PCR cycle.
4. Construct the plasmid
4.1 We design another kind of primer and reverse which contain EcoRI and Hind III site. The condition to amplify it by PCR is same as above all. We choose pET28a(+) as our expression vector. And then, we use enzymes of EcoRI site and XhoI site to digest the PCR product and plasmid. We use T4 DNA ligase to connect them at the temperature of 22 centigrade. We insert the plasmid into competent DH5α. It has been confirmed that these sequences we get are right when detecting them in company.
4.2 detail of the experiment
Digest (60μl) 1h
|
ddH2O |
31μl |
|
buffer |
6μl |
|
Fd1 |
1.5μl |
|
Fd2 |
1.5μl |
|
DNA |
20μl |
T4 DNA ligase to connect（20μl） 1h
|
Buffer |
2μl |
|
T4 DNA ligase |
0.5μl |
|
plasmid |
X |
|
fragment |
5X |
