Team:Hamilton McMaster/Methodologies

DNA

The majority of the DNA was provided by iGEM, either on the Plates 1-5 or by order. These included:

  • 1C3-pbla
  • T4 holin
  • T4 endolysin
  • cph8
  • PompC-GFP
  • pbad/araC
  • ho1
  • PcyA
  • 1A3 backbone
  • 1K3 backbone
  • 1C3 backbone
  • 1S3 backbone

The light sensing genes were isolated via PCR from a cassette purchased from Addgene. This cassette included the following genes:

  • Ccas
  • Ccar
  • Pcpcg2

PCR

Because the DNA from the cassette, when isolated via PCR, would not have the usable cut sites (E X, and S P), the primers were designed with overhangs. Annealing temperatures were calculated in two stages, using the Warren A. Kibbe OligoCalc. The first stage only included the section of the forward and reverse primers that connected to the DNA in the cassette. The second stage included the entire length of the primers, as the overhangs had been formed.

Ccar PCR Thermocycle:

  1. 95°C for 2min
  2. 95°C for 30sec
  3. 45°C for 30sec
  4. 72°C for 1min
  5. Repeat a-d for for 15 cycles
  6. 95°C for 30sec
  7. 56°C for 30sec
  8. 72°C for 1min
  9. Repeat f-i for 15 cycles
  10. 4°C indefinitely

Ccas PCR Thermocycle:

  1. 95°C for 2min
  2. 95°C for 30sec
  3. 45°C for 30sec
  4. 72°C for 2:30min
  5. Repeat a-d for for 15 cycles
  6. 95°C for 30sec
  7. 58°C for 30sec
  8. 72°C for 2:30min
  9. Repeat f-i for 15 cycles
  10. 4°C indefinitely

Pcpcg2 PCR Thermocycle:

  1. 95°C for 2min
  2. 95°C for 30sec
  3. 43°C for 30sec
  4. 72°C for 45sec
  5. Repeat a-d for for 15 cycles
  6. 95°C for 30sec
  7. 57°C for 30sec
  8. 72°C for 45sec
  9. Repeat f-i for 15 cycles
  10. 4°C indefinitely
Cells

E. coli DH5α cells (Life Technologies / Invitrogen) were used for the entire transformation process.

Buffers and Enzymes for Digestion and Ligation

Buffers and enzymes (New England Biolabs):

  • CutSmart
  • NEBuffer 3.1
  • EcoRI (E)
  • Xbal (X)
  • SpeI (S)
  • PstI (P)
  • T4 DNA ligase

Through parallel tests of all enzymes in all buffers, we found that CutSmart was the best buffer for all enzymatic digestions.

Mini-Prep

Mini-prep materials were bought from Life Technologies / Invitrogen. Mini-prep procedure followed standard protocol, involving centrifugation of cell samples into columns, and removal of supernatant. Mini-prep was followed by gel extraction.

 To accomplish this we will incorporate the theory behind multichromatic control of gene expression and the holin-endolysin system. The system will consist of three plasmids, a chromophore plasmid that contains the chromophore necessary for activation of light sensitive transcription factors so that the systems can be managed in the presence of light. The red plasmid will contain the protein of interest downstream of a promoter that is induced by red light and the green plasmid will contain the holin-endolysin dual system downstream of a promoter that is induced by green light.