Phase 1: PCR Amplification of Genes

From 2006.igem.org

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 (Xymo 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 Xymo 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.