IGEM:NYMU/2007/Construct

Assembly Ordering and Progress

system 1

System 1: glucose-sensing insulin generator (5 task left: 2 PCR and 3 biobrick standard assembly)

pOmpC

RBS + TAT

INSA

RBS + TAT

INSB

RBS

CinR

RBS

HSL

D-term

duration

members

assembly

status

RBS + CinR

RBS + HSL

8/30-8/31

翔

1st assembly

9/01, 07

CinR + HSL (RBS+)

9/1-9/4

宥,君,威

2nd assembly

9/02, 07

CinR+HSL(RBS+) + D-term

9/16-9/27

宥,君,翔,pett

3rd assembly

9/27 07

RBS + TATA+ INSA (PCR)

RBS + TATB+ INSB (PCR)

9/28-9/30

銘, pett

basic part (PCR)

9/30, 07

pOmpC + TATA_INSA (failed)

TATB_INSB + CinR+HSL+D-term (wrong assembly)

-10/5

pett

4th assembly

10/5,7,8 07

pOmpC + TATA_INSA

-10/11

pett

4th assembly

pOmpC + TATA_INS_A + TATB_INS_B

-10/23

4th assembly

pOmpC + TATA_INS_A + TATB_INS_B + CinR+HSL+D-term

Final

system 2

System 2: insulin-sensing insulin destoryer (5 tasks left: 2 PCR and 3 biobrick standard assembly)
pCinRHSL	OmpRBS	RBS + TATD	IDE	D-term	duration	members	assembly	status
	OmpRBS (PCR)	RBS + TATD+ IDE (PCR)			-102	銘, 茹, pett	basic part (PCR)	10/2 07
pCinRHSL + OmpRBS					-10/5	pett	1st assembly	10/5 07
pCinRHSL + OmpRBS + TATD_IDE (RBS+)					-10/11	pett	2nd assembly
pCinRHSL+OmpRBS+TATD_IDE + D-term							Final

Promoter assay construct: pOmpC+(RBS+EYFP)

our first contruct for promoter activity assay under glucose stimulus Insert size: 866bp (XbaI to SpeI) Total vector size: 3,019bp V-I: 2,153bp May be checked with: BtgZI 8/27, 2007

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OmpRBS assay construct: pLacIQ + OmpRBS + GFP

our first construct for OmpRBS blocking assay under glucose stimulus Insert size: 1004bp (XbaI to SpeI) Total vector size: 5,425bp V-I: 4,421bp May be checked with: BtgZI 10/24, 2007

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OmpRBS assay construct: pLacIQ + OmpRBS

our first construct for OmpRBS blocking assay under glucose stimulus Insert size: 120bp (XbaI to SpeI) Total vector size: 4,541bp V-I: 4,421bp May be checked with: 12/13, 2007

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System 1: pOmpC + (TATA+INSA) + (TATB+INSB) + (RBS+CinR) + (RBS+HSL) + D-term

Phase 1 of system 1: RBS + CinR

Insert size: 790bp Total vector size: 2,943bp V-I: 2,153bp May be checked with: BsaBI

Phase 1 of system 1: RBS + HSL

Insert size: 730bp Total vector size: 2,883bp V-I: 2,153bp May be checked with: AgeI

Phase 1 of system 1: TATA + INSA = TATA_INSA

Insert size: 170bp PCR product size: 209bp May be checked with: RsaI There is a extra SpeI site formed due to the addition of one extra stop codon

Phase 1 of system 1: TATB + INSB = TATB_INSB

Insert size: 220bp PCR product size: 233bp May be checked with AluI

Phase 2 of system 1: pOmpC + TATA_INSA

Insert size: 292bp Total vector size: 2,454bp V-I: 2,162bp

Phase 2 of system 1: (RBS+CinR) + (RBS+HSL) = CinR+HSL

Insert size: 1,524bp Total vector size: 3,677bp V-I: 2153bp

Phase 3 of system 1: pOmpC + TATA_INSA + TATB_INSB

Insert size: 502bp Total vector size: 2,655bp V-I: 2,153bp

Phase 3 of system 1: CinR+HSL + D-term = CinR+HSL+D-term

Insert size: 1,633bp Total vector size: 4,818bp V-I: 3,185bp

Phase 4 of system 1: pOmpC + TATA_INSA + TATB_INSB + CinR+HSL+D-term

Insert size: 2,139bp Total vector size: 4,292bp V-I: 2,153bp

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System 2: pCinRHSL+ OmpRBS + (TATD+IDE) + D-term

Phase 1 of system 2: TATD + IDE = TATD_IDE

Insert size: 3,190bp PCR product size: 3,203bp

Phase 1 of system 2: pCinRHSL + OmpRBS

Insert size: 308bp Total vector size: 2,461bp V-I: 2,153bp

Phase 2 of system 2: pCinRHSL + OmpRBS + TATD_IDE

Insert size: 3,488bp Total vector size: 5,641bp V-I: 2,153bp

Phase 3 of system 2: pCinRHSL + OmpRBS +  TATD_IDE + D-term

Insert size: 3,589bp Total vector size: 5,742bp V-I: 2,153bp

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Basic Constructs

pOmpC in pSB1A2

Insert size: 118bp Total vector size: 2,271bp V-I: 2,153bp

pCinRHSL in pSB1A2

Insert size: 235bp Total vector size: 2,388bp V-I: 2,153bp

RBS in pSB1A2

BseR1 can be applied to check the insertion of RBS Insert size: 22bp Total vector size: 2,175bp V-I: 2,153bp

D-term in pSB1AK3

Insert size: 105bp Total vector size: 3,290bp V-I: 3,185bp