IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/05/25

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Antibiotic Plates

Test on Kanamycin Plates

  • Kan plates worked well; growth observed on LB-only but not on LB-Kan.
  • Smear of growth observed on LB-only plate probably due to plates not drying well.

Results of Test on Chloramphenicol plates

  • Chloramphenicol plates did not work well; growth observed on both LB-Chlo plates and LB-only plates.
  • Could be due to aliquot of chloramphenicol not working at the desired concentrations.
  • Will test a different aliquot of chloramphenicol in the same fashion (spread-plated 1X, streaked ON culture); if that doesn`t work, then try growing in liquid media.
  • If that works, then next time we make LB-Chlo it will have to be dissolved in the liquid media before it is poured.
  • If liquid media doesn't work, then make a new batch of LB-Chlo plates.

Tested tetracycline

  • added 20μL of sd.H2O to LB plate
  • added 20μL of 15mg/mL tetracycline to LB plate (thus creating a 1x spreaded solution)
  • spreaded tetracycline evenly over LB plate
  • covered with aluminum foil (tetracycline is light sensitive)
  • sat opened plate on bench for 30 minutes to allow water to evaporate
  • streaked DB3.1 cells onto the LB-tet plate and a LB-only plate
  • put both plates into 37°C incubator in Lagally Lab BioHazard room for 1 day

Antibiotic Liquid Media

  • Made LB Broth+Amp (50µL Amp in 50mL LB Broth).
  • Stored on the shelf of Roza's (original) bench.

Transformation of Biobricks

  • The Amp Construction Plasmid plate showed growth of cell colonies
  • The Chloramphenicol Construction Plasmid plate had a smear growth pattern, possibly due to the plates not drying well
  • All other plates had no growth
  • These observed results may have been caused by low plasmid DNA concentrations, will do a test to determine the concentration of the resuspended Biobricks

Test of Resuspended Biobrick Concentrations

Biobrick Part Concentration (µg/µl) A260/280 A260/230
Amp Construction Plasmid63.960.841.63
Kan Construction Plasmid89.890.801.60
Terminator63.850.811.59
J23100/RBS71.651.002.10
pBAD65.470.841.58
Tet Construction Plasmid64.600.831.55
Chlo Construction Plasmid67.140.891.72
GFP62.470.841.64

Notes

  • 1 ul of resuspended biobrick was used with a Nanodrop 1000 to get the above results
  • The red dye in the biobrick solution may influence the purity readings by shifting the spectrum.
  • Spectral shift may also lower the measured DNA concentration compared to the actual DNA concentration.

Conclusions

  • Low transformation results may not have been due to concentration of DNA, as they were all more or less the same. Nonetheless, we think that for some parts, there may not have been enough DNA, so transformation done tomorrow would be with 2µL of DNA and not 1 µL.

Innoculation of Amp Construction Plasmid Cells

  • Since the Amp construction plasmid cells were successfully transformed, they were innoculated into 10mL of liquid LB+Amp and incubated in the AMBL 37ºC shaking incubator overnight, shaking at 100 rpm.


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