-Visualization-

@plasmid extraction oriTr-GFP-oriTr and JM109

@plasmid extraction and DNA refine oriTr-GFP-oriTr and JM109

@DNA densitometry

oriTr-GFP-oriTr 5 26.1 ng/μl

oriTr-GFP-oriTr 6 51.3 ng/μl

oriTr-GFP-oriTr 7 47.2 ng/μl

oriTr-GFP-oriTr 8 58.4 ng/μl

@restriction enzyme to oriTr-GFP-oriTr (XbaⅠ, PstⅠ)

@electrophoresis and gel extraction oriTr-GFP-oriTr5～8 and pSB1C3(restriction enzymed 9/15)

@DNA densitometry

totaled oriTr-GFP-oriTr5,6,8 18.4 ng/μl

pSB1C3① 16.5 ng/μl

pSB1C3② 11.5 ng/μl

@electrophoresis for confirmation, but could not find bind.

-Digestion

Promoter: Spe and Pst

Suppressed expression: Xba and Pst

-Electrophoresis

Above samples

-Gel extraction

Above samples

-Sequencing-

@Killer＋Target

no.1J23100-K317037-K283030　/　VF

could read 500 base which contain vector and the first half of J23100 and K317037, but not bond to no.2

no.2 J23100-K317037-K283030　/　VR

could read 500 base which contain vector and the latter half of K283030,

but could not bond to no.1, deficiene 50 base from terminator codon of CheZ in K283030 (also terminator codon).

no.3 P0140　/　VF

no.4 P0140　/　VR

could not find both of the sequence

@Visual

no.5 OriT_GFP_OriT_1　/　VF

could read about 350 base and confirmate vector and part of OriT for 60 base

no.6 OriT_GFP_OriT_1　/　GFP

no.7 OriT_GFP_OriT_1　/　VR

could read about 650 base from no.6 and about 350 base from no.7,

and bond each sequence to one

no.8 OriT_GFP_OriT_2　/　VF

could read about 350 base and confirmate vector and part of OriT for 60 base

no.9 OriT_GFP_OriT_2　/　GFP

could read about 600 base, but that is just sequence of GFP

no.10 OriT_GFP_OriT_2　/　VR

could read 300 base, but not agree with OriT_GFP_OriT

no.11 OriT_GFP_OriT_3　/　VF

could read 250 base which contain OriT for 60 base and vector for other sequence

no.12 OriT_GFP_OriT_3　/　GFP

could not read sequence

no.13 OriT_GFP_OriT_3　/　VR

could read about 300 base and find sequence of OriT

no.14 OriT_GFP_OriT_4　/　VF

could read 200 base and confirmated vector and the first half of OriT

no.15 OriT_GFP_OriT_4　/　GFP

could read about 600 base, but that is just sequence of GFP

no.16 OriT_GFP_OriT_4　/　VR

could read about 300 base and confirmated the latter half of OriT

@summary

Targeting

succeed in ligation, but deficiene 50 base from terminator codon of CheZ in K28303.

I guessed there is not His-tag.

could not read sequence of P0140.

decrease tenpate concentration and sequencing again

Visual

4 sumples of OriT-GFP-OriT was turned out wrong.

1 and 4 were OriT-GFP.

2 was unknown.

3 was OriT only, which is self ligation.

-Targetting-

@preculture P0140, J23100-K317037-K283030(Targetting Device), pSB1C3

@plasmid extraction and DNA refine P0140, J23100-K317037-K283030(Targeting Device), pSB1C3

@restriction enzyme(XbaⅠ, Pst) P0140, J23100-K317037-K283030(Targeting Device), pSB1C3