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Team:Kyoto/Notebook
From 2012.igem.org
Contents |
Secretion
Florigen
August 2
Mutation of FT by Sato
FT gene has two BioBrick restriction enzyme sites, EcoR1 and Pst1 which is next to each other.
So we tried to delete both at once by using two primers with mutation.
| 10xBufer | 2mM dNTPs | primer fwd | primer rev | template | polymerase | MilliQ | Total |
|---|---|---|---|---|---|---|---|
| 5 | 5 | 1.5 | 1.5 | 0.5 | 1 | 35.5 | 50 |
94°C 2min, (98°C 10sec, 68°C 4min)x4cycles, 4°C Hold
| PCR product | Dpn1 |
|---|---|
| 50 | 2 |
| product | MilliQ | Ligase | T4 Kinase | Total |
|---|---|---|---|---|
| 2 | 7 | 5 | 1 | 15 |
16°C, 1h incubate
| competent cell | DNA |
|---|---|
| 20 | 2 |
Cells were stored on ice for 30min.
After 42°C 60sec heat shock, cells were stored on ice for 2min.
Then cells were precultureed at 37°C for 1hr, plated to Kanamycin plate.
August 13
Liquid culture of FT 37°C, overnight
August 14
Miniprep of FT : by Sato, Takeuchi
The concentrarion was 81.5ng/uL
Restriction digestion and Electrophoresis : by Sato
To check wheter mutation was succeed, we did restriction enzyme digestion.
| DNA(FT,80ng/uL) | 10xBuferH | EcoR1 | Pst1 | MilliQ | Total |
|---|---|---|---|---|---|
| 5 | 2 | 1 | - | 12 | 20 |
| 5 | 2 | - | 1 | 12 | 20 |
37°C 2h incubate
We did electrophoresis of three FT plasmids, non-cutted, EcoR1 cutted, Pst1 cutted.
However, we couldn't get any bands (data not shown.)
Liquid culture of FT (4mL)
August 15
Miniprep of FT : by Sato, Takeuchi, Hyungcheol
We couldn't get enough concentration of plasmids.
Electrophoresis : by Sato
We retried electrophoresis of three samples same as yesterday.
However, we couldn't get any bands as well.
August 16
Transformation : by Takeuchi, Ota
| Name | Well | Sample | Competent Cells | Total | Plate | Colony |
|---|---|---|---|---|---|---|
| FT | - | 1 µL | 10 | 11 | LB (Kan+) | × |
| pSB1C3 | 1-3-A | 1 | 10 | 11 | LB (CP+) | ○ |
| I719005 | 1-15-N | 1 | 10 | 11 | LB (Amp+) | ○ |
August 17
Transformation : by Takeuchi
| Name | Well | Sample | Competent Cells | MilliQ | Total | Plate | Colony |
|---|---|---|---|---|---|---|---|
| FT | - | 2 | 2 | 18 | 22 | LB (Kan+) | × |
We found that mutation of FT was not successful.
August 20
We decided to do PCR using FT specific primers before mutation.
PCR of FT : by Sato
| 10xBufer | dNTPs | MgSO4 | primer fwd | primer rev | template | polymerase | MilliQ | Total |
|---|---|---|---|---|---|---|---|---|
| 5 | 5 | 3 | 1 | 1 | 1(130ng/µL) | 1(KOD plus neo) | 33 | 50 |
94°C 2min, (98°C 10sec, 68°C 15sec)x30cycles, 4°C Hold
After the ethanol precipitation, we diluted in 30µL of MilliQ.
The concentration was 203ng/µL.
| Lane | Name | length(bp) |
|---|---|---|
| 1 | 1kb ladder | - |
| 2 | FT | 600 |
August 21
Restriction digestion : by Sato
| DNA(FT,203ng/µL) | 10xBuferM | Xba11 | Pst1 | MilliQ | Total |
|---|---|---|---|---|---|
| 10 | 4 | 1 | 1 | 24 | 40 |
37°C, 5h incubate
After the ethanol precipitation, we diluted in 30µL of MilliQ.
The concentration was 54,9ng/µL.
Ligation : by Sato
| Vector | Insert | Ligation High Ver.2 | ||
|---|---|---|---|---|
| pSB1C3 | 1 | FT | 10 | 5.5 |
Liquid culture
T7 promoter, pSB1C3 (4mL)
August 22
Miniprep : by Sato
| T7 promoter | pSB1C3 |
|---|---|
| 85.3ng/µL | 82.93ng/µL |
August 23
Ethanol Precipitation
diluted in 20µL 79.3ng/µL
mada dekite nai
August 24
Restriction enzyme processing
| T7 promoter(85.3ng/µL) | Spel | Pstl | buffer M | MiliQ | Total |
|---|---|---|---|---|---|
| 10 | 1 | 1 | 2 | 6 | 20 |
->purifying column 33.4ng/µL(dissolution 40µL)
| pSB1C3(82.9ng/µL) | Xbal | Spel | buffer M | MiliQ | Total |
|---|---|---|---|---|---|
| 20 | 1 | 1 | 4 | 14 | 40 |
->gene clean2 39.9ng/µL(dissolution 40µL)
<<picture1,2>>
Ligation
| FT(600bp, 79.3ng/µL) | pSB1C3(2000bp,39.9ng/µL) | Ligation High Ver.2 |
|---|---|---|
| 3µL => 597fmol | 2µL => 60fmol | 2.5µL |
| FT(600bp, 79.3ng/µL) | T7(2100bp,33.4ng/µL) | Ligation High Ver.2 |
|---|---|---|
| 2.4µL => 478fmol | 2µL => 48fmol | 2.2µL |
=> 16℃,1hr incubate
August 27
Colony PCR
| 2X Quick Tag | VF2 | VR | MiliQ | Total |
|---|---|---|---|---|
| 25 | 1 | 1 | 23 | 50 |
Lane1: 1kb ladder
Lane2~16: FT(pSB1C3) about 800bp
FT(TOPO) PCR(re)
| buffer | dNTPs | MgSO4 | primer f | primer r | Template(130ng/µL) | KODplus neo | MilliQ | Total |
|---|---|---|---|---|---|---|---|---|
| 5 | 5 | 3 | 1 | 1 | 1 | 1 | 33 | 50 |
| 94℃ | 98℃ | 68℃ | cycle |
|---|---|---|---|
| 2min | 10sec | 15sec | 30 |
Liquid culture (FT) 4ml by Nobeyama
August 28
Mutaion of FT (re)
inverse PCR
| MilliQ | buffer | dNTP | primer f | primer r | FT(130ng/µL) | KODplus | Total |
|---|---|---|---|---|---|---|---|
| 35.5 | 5 | 5 | 1.5 | 1.5 | 0.5 | 1 | 50 |
| 94℃ | 98℃ | 68℃ | cycle |
|---|---|---|---|
| 2min | 10sec | 4sec | 18 |
Lane1: 1kb ladder
Lane2: FT
Miniprep FT(TOPO)
158ng/µL
Tranformation
competent cell: 20
BBa.I746902 : 2
(plate 316f)
(GFP generator of pBAD/araC-mut3 GFP:6His-DT)
Golden Gate Assembly
August 10
Golden Gate Assembly method This method helps us to constract some genes quickly and we can design the order of constractions. We are trying to construct a gene cycle composed of 6 genes( pSB1A3, lacI, GFP, RFP, GFP, DT). After this experiment is confirmed to work, we will so some experiment to check how the numbers of promorters influences the strength of transcription.
