Phase 1: PCR Amplification of Genes
From 2006.igem.org
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::*Notes: | ::*Notes: | ||
:::Amplification of ComX alone was unsuccessful. However, ComQ and ComX are adjacent on the genome so we subsequently amplified them together. | :::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 | + | :::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 | : 2. ComP: 2.3kb, B. subtilis membrane receptor for ComX pheromone | ||
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:::Rev = GACTGCAGCTACTAGTTACAATTCGATTTCAATATCAGCCTTAAAGCC (Tm = 70.71 F) | :::Rev = GACTGCAGCTACTAGTTACAATTCGATTTCAATATCAGCCTTAAAGCC (Tm = 70.71 F) | ||
::*Notes: | ::*Notes: | ||
| - | :::Difficult to obtain sufficient quantity. DNA concentration by | + | :::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. | : 3. ComA: 645kb, transcription factor activated by a ligand-bound ComP. | ||
Latest revision as of 21:19, 31 October 2006
A. Isolating necessary genes for the chemotaxis mechanism from genomic E. coli DNA
- 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.
