Team:IIT Madras/Project

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Contents

Project Details

Part Design

PCR Products

Legend
  • This color represents the portion to be added by Extension PCR / Inverse PCR / Assembly PCR.
  • This color represents sites that lie in the middle of the parts to be submitted. These are placed so that the promoters and the inserts can be switched out.
  • This represents an apparent restriction site, one that cannot be used due to the sequences' presence in an ORF
SCM
Gene of 295 bp to be synthesised by Assembly PCR / LCR.
Block 1
  1. Kpn1 | P170 | RBS | HindIII
  2. Kpn1 | NICE | RBS | HindIII
  3. HindIII| GFP | Terminator | BamH1
  4. Kpn1| Constitutive | RBS | GFP | Terminator | BamH1
Block 2
  1. Kpn1 | Constitutive | RBS | HindIII | SP310mut2 | Nhe1 | GFP | Terminator | BamH1 (by Inverse PCR) // Can't design a Primer this big.
  2. Kpn1 | Constitutive | RBS | HindIII | SP310mut2 | Nhe1
  3. Nhe1 | GFP | Terminator | BamH1 (Extension PCR from 1.4)
Block 3
  1. Kpn1 | Constitutive | RBS | HindIII
  2. HindIII | CRE-NLS | Terminator | Sac1
  3. Sac1 | Constitutive | RBS | loxP | Nhe1 | GFP | Terminator | loxP | Xho1 (by Inverse PCR + Extension PCR)
  4. Xho1 | RFP | Terminator | BamH1
Final Design
  1. Kpn1 | NICE | RBS | HindIII (as made in 1.1)
  2. HindIII | CRE-NLS | Terminator | Sac1 (as made in 3.2)
  3. Sac1 | loxP | P170 | RBS | SP310mut2 | Xho1 (by Inverse PCR + Extension PCR)
  4. Xho1 | SCM | Nhe1 | loxP | Terminator | BamH1
Part Submission
  1. EcoR1 | Not1 | Xba1 | NICE | RBS | Spe1 | Not1 | Pst1
  2. EcoR1 | Not1 | Xba1 | P170 | RBS | Spe1 | Not1 | Pst1
  3. EcoR1 | Not1 | Xba1 | Kpn1 | Constitutive | RBS | HindIII | SP310mut2 | Nhe1 | GFP | Terminator | BamH1 | Spe1 | Not1 | Pst1
  4. EcoR1 | Not1 | Xba1 | Kpn1 | Constitutive | RBS | HindIII | CRE | Terminator | Sac1 | Constitutive | RBS | loxP | Sma1 | GFP | Terminator | loxP | Xho1 | RFP | Terminator | BamH1 | Spe1 | Not1 | Xba1
  5. EcoR1 | Not1 | Xba1 | Kpn1 | NICE | RBS | HindIII | CRE-NLS | Terminator | Sac1 | loxP | P170 | RBS | SP310mut2 | Xho1 | SCM | BamH1 | loxP | Terminator | Spe1 | Not1 | Pst1

Proof of Concept

Block 1 - To characterize promoters against a standard
  • P170 | GFP | Terminator
  • NICE | GFP | Terminator
  • Constitutive | GFP | Terminator
Block 2 - Test of export efficiency
  • Constitutive | GFP | Terminator
  • Constitutive | SP310mut2 | GFP | Terminator
Block 3 -To test CRE-loxP system
  • Constitutive | RBS | CRE | Terminator | Constitutive | RBS | loxP | GFP | Terminator | loxP | RFP | Terminator
Acid Tolerance Promoter (P170) | CRE | Terminator | loxP | Consitutive | GFP | Terminator | loxP | RFP | Terminator

Final design

NICE | CRE | Terminator | loxP | Acid Tolerance Inducer (P170) | Export tag | SCM | loxP | Terminator

The Experiments

Part 3

Results

 

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