Part:BBa_K1313004

Neomycin Resistance

Neomycin resistance gene. This gene can be used for selection of cells with an efficient transformation. This gene codes neomycin phosphotransferase which breaks neomycin, kanamycin, and gentamicin molecules. If you use these gene as selectivity gene in bacteria, and your laboratory lacks neomycin, you can replace it for kanamycin by adding 35 mg/ml per liter of LB media. The best concentration of neomycin to use is 80 mg/ml per liter of LB media.

This gene can be also used as a selective gene for mammalian cells using gentamicin.

(BBa_K1313004)

Aim

To characterize the part responsible for neomycin resistance (BBa_K1313004) and its response to kanamycin in DH5 alpha E. coli cells using OD measurements at 600 nm in the presence or absence of a plasmid containing the resistance sequence.

Experimental design

Preparation:

         PLBDE2 plasmid was used, however due to it being too big, a digestion was done followed by a ligation. The transformation had been concluded successful. However, the experiments with those cells were only successful if they were done with a double overnight culture.

Experiment

           Bacteria with neomycin resistance and bacteria without such have been inoculated overnight.

8 vials were set up with different kanamycin concentrations ranging from 0 µg/µL to 200 µg/µL. In each one were added 100 µL of DH5alphas with neomycin resistance cassette. Another 8 vials with different kanamycin concentrations were prepared and 100 µL of non-resistant DH5 alphas were added in each vial. OD measurements at 600 nm were taken every 30 minutes to follow colony growth for 4 hours.

Experiment was repeated with higher kanamycin concentrations ranging from 0 µg/µL to 1000 µg/µL and the same observation as the previous experiment could be concluded.

Observations

• Cells with resistance grew overtime, and the vials containing high kanamycin concentrations produced slower cell growth
• Cells without resistance grew only in the vial with 0 µg/ µL kanamycin concentration
• We can offer no explanation why the experiment was successful with only double overnight cultures

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 165
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 165
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 165
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 165
  Illegal NgoMIV site found at 616
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 465
  Illegal SapI.rc site found at 675