Template:BioE140LSpr09-Growth

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 (see Transformation section below)

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 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.

Transformation

Thaw 2 tubes of 280 uL aliquot of cells on ice

Add 100 uL of water to each tube

Add 60 uL of KCM salts to each tube

Add 1ul of the constructs to 20 uL of the cell cocktail. Pipette up and down gently to mix

Let sit on ice for 10 min

Heat shock for 2 min at 42

Put back on ice for 1 min

Add 100uL of LB, let shake in the 37 degree incubator for 40 min

Plate on chloramphenicol/ampicillin selective plates, let incubate overnight

Results

Construct 11 --> M10210 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm}
Construct 12 --> M10211 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm}
Construct 13 --> M10212 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm}
Construct 14 --> M10213 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm}
Construct 15 --> M10214 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm
Construct 16 --> M10215 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm}
Construct 17 --> M10216 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm}
Construct 18 --> M10217 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<TshA!}{dblTerm}
Construct 19 --> M10218 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPG_L6!}{dblTerm}
Construct 20 --> M10219 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<eCPX!}{dblTerm}
Construct 21 --> M10220 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm}
Construct 22 --> M10221 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<Ag43_short!}{dblTerm}
Construct 23 --> M10222 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm}
Construct 24 --> M10223 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm}
Construct 25 --> M10224 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPompX!}{dblTerm}
Construct 26 --> M10225 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<TshA!}{dblTerm}

Controls are DH10B and pBca9495CA-Bca1144

http://openwetware.org/images/0/09/Bioe140L-GrowthPlotsConstr.pdf
http://openwetware.org/wiki/Image:Bioe140L-GrowthPlotsConstr.pdf

Analysis

Construct 11 --> M10210 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm}
Construct 12 --> M10211 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm}
Construct 13 --> M10212 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm}
Construct 14 --> M10213 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm}
Construct 15 --> M10214 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm
Construct 16 --> M10215 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm}
Construct 17 --> M10216 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm}
Construct 18 --> M10217 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<TshA!}{dblTerm}
Construct 19 --> M10218 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPG_L6!}{dblTerm}
Construct 20 --> M10219 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<eCPX!}{dblTerm}
Construct 21 --> M10220 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm}
Construct 22 --> M10221 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<Ag43_short!}{dblTerm}
Construct 23 --> M10222 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm}
Construct 24 --> M10223 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm}
Construct 25 --> M10224 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPompX!}{dblTerm}
Construct 26 --> M10225 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<TshA!}{dblTerm}

Controls are DH10B and pBca9495CA-Bca1144

http://openwetware.org/images/5/5f/BioE140L-GrowthRatesConstr.pdf
http://openwetware.org/wiki/Image:BioE140L-GrowthRatesConstr.pdf

http://openwetware.org/images/4/4b/BioE140L-LogFit.pdf
http://openwetware.org/wiki/Image:BioE140L-LogFit.pdf