IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/08/04

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Testing of Lock function with Reporters

assembled assemblies 16 to 21

1. Digestion using standard UBC iGEM digestion protocol

  • Prefix samples (cut with EcoRI and SpeI)
  • BBa_J23100
  • BBa_E0022
  • BBa_E1010
  • BBa_E0030
  • K145015
  • Suffix samples (cut with XbaI and PstI)
  • lock 1
  • BBa_B0014
  • BBa_B0034

  • Backbone cut (cut with PstI and EcoRI)


2. Ligation

using standard UBC iGEM digestion protocol, volumes calculated and optimized by the UBC iGEM j to i calculator

  • ligation into pSB1C3':
  • ASEM 16: BBa_J23100 + Lock1
  • ASEM 17: BBa_E0022 + BBa_B0014
  • ASEM18: BBa_E1010 + BBa_B0014
  • ASEM 19: BBa_E0040 + BBa_B0014
  • ASEM 20: BBa_K145015 + BBa_B0014
  • ASEM 21: BBa_J23100 + BBa_B0034

Characterizing of RFP in M9 Minimal Media

  • Start cultures of pBADweak+GFP, pBADwt+GFP,pBADweak+RFP, pBADwt+RFP, pBADstrong+RFP, BW27783 and J23100 in 3mL M9 Minimal Media + associated antibiotic from streak plates

Transformation of J23101, J23105, J23100

  • In order to characterize pBAD relative to standard promoters, first need to standardize the curve of constituant promoters. BW27783 is used to characterize pBAD because it should have uniform arabinose induction. However, previous experiments using relative promoter activity have been done in TOP10 cells. In order to see the difference between BW27783 cells and TOP10 cells, first need to establish a curve using the constituant promoters that have previously been characterized.
  • Transform the three constituant promoters J23100, J23105 and J23100 into DH5a and BW27783 to characterize the growth.
  • All transformations were performed using the iGEM:British Columbia Transformation protocol using 1uL of 10ng/uL plasmid. All transformations were plated on ampicillin.