IGEM:Indian Institue of Technology Madras/2009/Notebook/PLASMID - Plasmid Locking Assembly for Sustaining Multiple Insert DNA/2009/10/15

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  • Checked the inoculums of the cotransformed cells under a fluorescence microscope
  • Not all the samples showed both the fluorescences.
  • only 3 samples - C1A R2212, C1A R1212 and C1A R1122- had both red and cyan glowing cells. Even in those samples, the intensity of the cyan was about 10 fold lesser than the intensity of red fluorescence. Also not all cells had both the fluorescence. This could in essence mean that the plasmid loss might be working.
  • The actual measurements of the plasmid loss will be conducted in 2 stages.
  • first stage - Each of DH5a and K272002 in pSB1A2 cells will be grown in 4 different media conditions - LB broth with no antibiotic, LB broth containing 25ug/ul ampicillin, LB broth containing 25ug/ul chloramphenicol and LB broth containing 25ug/ul of both amp and chl.
  • second stage - K272001 in pSB1C3 cells and K272001(1C3)-K272002(1A2) cotransformated cells will be grown in 4 different media condititions - LB broth with no antibiotic, LB broth containing 25ug/ul ampicillin, LB broth containing 25ug/ul chloramphenicol and LB broth containing 25ug/ul of both amp and chl.
  • Prep for the first stage of experiments
  • To check approximately at what time the inoculated cultures hit OD in between 0.1 and 0.5
  • {0.5/(OD-0.1)} ml of the inoculum should be transfered into each of the 4 tubes for the first stage of the experiments (this is approximately 0.01 OD to start with)
  • For the prep phase, 4 tubes of 3 ml LB (no AB) have been inoculted with DH5a, 4 tubes of 3ml LB (25ug/ml) have been inoculated with CFP in 1A2 and 4 tubes of 3ml LB (50ug/ml) have been inoculated with CFP in 1A2.
  • The OD of the samples was measured after 3.5 hours
  • DH5a-1 - 0.253
  • DH5a-2 - 0.091
  • DH5a-3 - 0.66
  • DH5a-4 - 0.178
  • CA-25-1 - 0.77 (that is CFP in 1A2, grown in LB containing 25ug/ml)
  • CA-25-2 - 0.221
  • CA-25-3 - 0.151
  • CA-25-4 - 0.086
  • CA-50-1 - 0.082 (that is CFP in 1A2, grown in LB containing 50ug/ml)
  • CA-50-2 - 0.120
  • CA-50-3 - 0.130
  • CA-50-4 - 0.056
  • this implies about 4 hours of initial incubation should be enough to reach an OD between 0.1 - 0.5, which can then be used to inoculte the final measurements media.