Team:Kyoto/Notebook

From 2012.igem.org

Revision as of 10:11, 5 September 2012 by Hyungcheol (Talk | contribs)

  • Home
  • Project
  • Method And Material
  • Notebook
  • Consideration
  • Team
  • SiteMap

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.

Inverse PCR
10xBufer2mM dNTPsprimer fwdprimer revtemplatepolymeraseMilliQTotal
551.51.50.5135.550

94°C 2min, (98°C 10sec, 68°C 4min)x4cycles, 4°C Hold

Dpn1 Digestion
PCR productDpn1
502
37°C,1h incubate
Self-ligation
productMilliQLigaseT4 KinaseTotal
275115

16°C, 1h incubate

Transformation
competent cellDNA
202

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)10xBuferHEcoR1Pst1MilliQTotal
521-1220
52-11220

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

NameWellSampleCompetent CellsTotalPlateColony
FT-1 µL1011LB (Kan+)×
pSB1C31-3-A11011LB (CP+)
I7190051-15-N11011LB (Amp+)

August 17

Transformation : by Takeuchi

NameWellSampleCompetent CellsMilliQTotalPlateColony
FT-221822LB (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

10xBuferdNTPsMgSO4primer fwdprimer revtemplatepolymeraseMilliQTotal
553111(130ng/µL)1(KOD plus neo)3350

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.

Electrophoresis
Electrophoresis0821.png
LaneNamelength(bp)
11kb ladder-
2FT600


August 21

Restriction digestion  : by Sato

DNA(FT,203ng/µL)10xBuferMXba11Pst1MilliQTotal
104112440

37°C, 5h incubate
After the ethanol precipitation, we diluted in 30µL of MilliQ.
The concentration was 54,9ng/µL.

Ligation  : by Sato

VectorInsertLigation High Ver.2
pSB1C31FT105.5

Liquid culture
T7 promoter, pSB1C3 (4mL)

August 22

Miniprep : by Sato

T7 promoterpSB1C3
85.3ng/µL82.93ng/µL


August 23

Ethanol Precipitation
diluted in 20µL 79.3ng/µL

mada dekite nai


August 24

Restriction enzyme processing


T7 promoter(85.3ng/µL)SpelPstlbuffer MMiliQTotal
10112620


->purifying column 33.4ng/µL(dissolution 40µL)


pSB1C3(82.9ng/µL)XbalSpelbuffer MMiliQTotal
201141440


->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 => 597fmol2µL => 60fmol2.5µL

FT(600bp, 79.3ng/µL)T7(2100bp,33.4ng/µL)Ligation High Ver.2
2.4µL => 478fmol2µL => 48fmol2.2µL


=> 16℃,1hr incubate

August 27

Colony PCR


2X Quick TagVF2VRMiliQTotal
25112350


Lane1: 1kb ladder
Lane2~16: FT(pSB1C3) about 800bp

FT(TOPO) PCR(re)

bufferdNTPsMgSO4primer fprimer rTemplate(130ng/µL)KODplus neoMilliQTotal
55311113350

94℃98℃68℃cycle
2min10sec15sec30

Liquid culture (FT) 4ml by Nobeyama

August 28

Mutaion of FT (re)

inverse PCR

MilliQbufferdNTPprimer fprimer rFT(130ng/µL)KODplusTotal
35.5551.51.50.5150
94℃98℃68℃cycle
2min10sec4sec18

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.