IGEM:NYMU/2007/Construct

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Contents

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 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

, 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)

<img src="http://parts.mit.edu/igem07/index.php/Image:POmpC-EYFP_%28plasmid%29.jpg">
  • 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

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

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

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

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

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
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