Template:BioE140LSpr09-Growth

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Growth Curve Assay

The purpose of this assay is to determine the toxicity of various composite constructs. These constructs include: 

M10210	{Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm}
M10211	{Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm}
M10212	{Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm}
M10213	{Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm}
M10214	{Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm}
M10215	{Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm}
M10216	{Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm}
M10217	{Pbad.rbs.prepro.StrepTag}{<AG4>}{<TshA!}{dblTerm}
M10218	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPG_L6!}{dblTerm}
M10219	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<eCPX!}{dblTerm}
M10220	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm}
M10221	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<Ag43_short!}{dblTerm}
M10222	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm}
M10223	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm}
M10224	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPompX!}{dblTerm}
M10225	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<TshA!}{dblTerm}

With all the constructs under an arabinose promoter, toxicity can be inferred from the differences in growth rate (as determined by changes in OD 600 over time) between samples containing LB and LB+arabinose.

Procedures

Plating

The controls are: DH10B, and pBca9495CA-Bca1144 (as controls, the DH10B is blank control and pBca9495CA-Bca1144 controls for plasmid effects.)

  • Take 2 tubes of 280ul of cell and add 60ul KCM and 100ul water to each.
  • Add 20ul of the KCM/water/cell solution into each construct.
  • Transform (heat shock, etc.)
  • Grow plates overnight.
  • Pick 5 colonies from each sample.
  • Grow to saturation in 96 well blocks with 400 uL LB media in each well.
  • Then from each of the 5 unique liquid cultures, make an arabinose sample and a non-arabinose sample.
  • Add 50 uL of LB media or LB Media+100ug/mL arabinose See note 1 below per well in 384 well plate.
  • Add 1 uL of cell sample to each well.
  • Place plate in Tecan and run OD measurements every 10 minutes.

Results

Analysis