Phase 1: PCR Amplification of Genes

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'''A.  Isolating genes for the quorum sensing mechanism from genomic ''B. subtilis'' DNA'''  
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'''A.  Isolating necessary genes for the chemotaxis mechanism from genomic ''E. coli'' DNA'''
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#ComQX: ComQ = 900bp, required accesory protein for ComX production.  ComX = 168bp, pheromone  
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#Tsr: 1.66kb, MCP I serine receptor 
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::*Primers: 
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:::Fwd = CGGAATTCGCTCTAGATGTTAAAACGTATCAAAATTGTGACCAGC (Tm = 70.19 deg F)
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:::Rev = GACTGCAGCTACTAGTTAAAATGTTTCCCAGTTCTCCTCG (Tm = 70.85 F)
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::*Notes:
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:::Relatively easy to obtain.
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:::Forward primer contains EcoRI and XbaI cut sites; Reverse primer contains SpeI and PstI cut sites.  [Similar to all primers]
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'''B.  Isolating genes for the quorum sensing mechanism from genomic ''B. subtilis'' DNA'''  
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: 1. ComQX: ComQ = 900bp, required accesory protein for ComX production.  ComX = 168bp, pheromone  
::*Primers:   
::*Primers:   
:::Fwd = CGGAATTCCCTCTAGATGAAGGAGATTGTGGAGCAAACATATTTAACG
:::Fwd = CGGAATTCCCTCTAGATGAAGGAGATTGTGGAGCAAACATATTTAACG
:::Rev = GACTGCAGCTACTAGTTAATCACCCCATTGACGGGTTATTGG
:::Rev = GACTGCAGCTACTAGTTAATCACCCCATTGACGGGTTATTGG
::*Notes:
::*Notes:
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:::Forward primer contains EcoRI and XbaI cut sites; Reverse primer contains SpeI and PstI cut sites
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:::Amplification of ComX alone was unsuccessful.  However, ComQ and ComX are adjacent on the genome so we subsequently amplified them together.
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:::Amplification of ComX alone was unsuccessful.  However, ComQ and ComX are adjacent on the genome so we subsequently amplified them together
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:::Series of DNA gel purifications Zymo extraction) and PCR amplifications necessary to obtain sufficient quantity.
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:::Series of DNA gel purifications (Xymo extraction) and PCR amplifications necessary to obtain sufficient quantity.
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: 2. ComP: 2.3kb, B. subtilis membrane receptor for ComX pheromone
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::*Primers: 
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:::Fwd = CGGAATTCCCTCTAGATGAAGAACTTAATAAAAAAATTCACAATAGCTG  (Tm = 68.08 deg F)
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:::Rev = GACTGCAGCTACTAGTTACAATTCGATTTCAATATCAGCCTTAAAGCC  (Tm = 70.71 F)
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::*Notes:
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:::Difficult to obtain sufficient quantity.  DNA concentration by Zymo extraction following PCR amplifications, then purification from gel was necessary to obtain sufficient quantity.
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: 3. ComA: 645kb, transcription factor activated by a ligand-bound ComP. 
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::*Primers: 
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:::Fwd = CGGAATTCCCTCTAGATGAAAAAGATACTAGTGATTGATGACCATCCGG  (Tm = 72.27 F)
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:::Rev = GACTGCAGCTACTAGTTAAAGTACACCGTCTGATTTCGCAATC  (Tm = 71.39 F)
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::*Notes:
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:::Relatively easy to obtain.
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'''C.  Additional prep work'''
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::*Isolate tet promoter, RBS, and tetR from the BioBrick Libraries.
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<br>
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<br>
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[[Rice University 2006|Back to Rice iGEM]] 
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<br>
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[[Phase 2: Plasmid Construction and Bacterial Transformations|On to Phase 2!]]

Latest revision as of 21:19, 31 October 2006

A. Isolating necessary genes for the chemotaxis mechanism from genomic E. coli DNA

  1. Tsr: 1.66kb, MCP I serine receptor
  • Primers:
Fwd = CGGAATTCGCTCTAGATGTTAAAACGTATCAAAATTGTGACCAGC (Tm = 70.19 deg F)
Rev = GACTGCAGCTACTAGTTAAAATGTTTCCCAGTTCTCCTCG (Tm = 70.85 F)
  • Notes:
Relatively easy to obtain.
Forward primer contains EcoRI and XbaI cut sites; Reverse primer contains SpeI and PstI cut sites. [Similar to all primers]

B. Isolating genes for the quorum sensing mechanism from genomic B. subtilis DNA

1. ComQX: ComQ = 900bp, required accesory protein for ComX production. ComX = 168bp, pheromone
  • Primers:
Fwd = CGGAATTCCCTCTAGATGAAGGAGATTGTGGAGCAAACATATTTAACG
Rev = GACTGCAGCTACTAGTTAATCACCCCATTGACGGGTTATTGG
  • Notes:
Amplification of ComX alone was unsuccessful. However, ComQ and ComX are adjacent on the genome so we subsequently amplified them together.
Series of DNA gel purifications Zymo extraction) and PCR amplifications necessary to obtain sufficient quantity.
2. ComP: 2.3kb, B. subtilis membrane receptor for ComX pheromone
  • Primers:
Fwd = CGGAATTCCCTCTAGATGAAGAACTTAATAAAAAAATTCACAATAGCTG (Tm = 68.08 deg F)
Rev = GACTGCAGCTACTAGTTACAATTCGATTTCAATATCAGCCTTAAAGCC (Tm = 70.71 F)
  • Notes:
Difficult to obtain sufficient quantity. DNA concentration by Zymo extraction following PCR amplifications, then purification from gel was necessary to obtain sufficient quantity.
3. ComA: 645kb, transcription factor activated by a ligand-bound ComP.
  • Primers:
Fwd = CGGAATTCCCTCTAGATGAAAAAGATACTAGTGATTGATGACCATCCGG (Tm = 72.27 F)
Rev = GACTGCAGCTACTAGTTAAAGTACACCGTCTGATTTCGCAATC (Tm = 71.39 F)
  • Notes:
Relatively easy to obtain.

C. Additional prep work

  • Isolate tet promoter, RBS, and tetR from the BioBrick Libraries.



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On to Phase 2!

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