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From 2012.igem.org

Contents 1 August 2 2 August 13 3 August 14 3.1 Miniprep of FT 3.2 Restriction digestion and Electrophoresis 4 August 10

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

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

Dpn1 Digestion

PCR product	Dpn1
50	2

37°C,1h incubate

Self-ligation

product	MilliQ	Ligase	T4 Kinase	Total
2	7	5	1	15

16°C, 1h incubate

Transformation

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

The concentrarion was 81.5ng/uL

Restriction digestion and Electrophoresis

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.)

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.

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