Device

Part:BBa_M10221

Designed by: Sadao Ota   Group: Berkeley BioE140L - S09   (2009-05-10)


{Pbad.rbs.prepro.StrepTag}}{˂GS5-IILK˃}{˂Ag43_short!}{dblTerm}

Composite part with an Arabinose promoter {Pbad}, ribosome binding site {rbs}, prepro periplasmic targetting sequence {prepro}, a streptavidin-binding tag {StrepTag}, a passenger part {GS5-IILK}, Autototansporter {Ag43 short!} which functions as the carrier protein for display of AG4 on the cell surface of E.coli, and a terminator {dblTerm}.

Template:BioE140LSpr09-AssayOverview


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


Silver Binding Assay

Silver Binding Assay – Characterization of AG4 composite parts

Information

Control: pBca9145-Bca1363

Composite Parts: 
11: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm}<br>
12: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm}<br>
13: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm}<br>
14: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm}<br>
15: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm}<br>
16: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm}<br>
17: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm}<br>

LB: ampicillin and kanamycin

Conditions: pH 7.4 TBS. Ambient light. Constant agitation. Silver Nitrate (AgNO3) does not react with LB or TBS at dilute concentrations.

Protocol

Pick and Incubate Colonies

1. Add 4mL of LB media with the appropriate antibiotics and arabinose
2. Pick a well-isolated, round, and "normal" looking colony containing the silver binding peptide with a pipet tip
3. Drop it in the test tube
4. Incubate at 37C overnight

Wash cells and incubate in AgNO3

5. Pellet 1 mL of saturated culture by spinning full speed, 30 seconds.
6. Dump supernatant, repeat to pellet another 1 mL (for a total of 2 mL)
7. Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
8. Add 200uL of TBS (pH 7.4) and resuspend
9. Centrifuge the colony solution for 30 seconds
10. Pour out the supernatant
11. Add TBS and 0.1mM AgNO3, and resuspend
12. Incubate at room temperature for 24hours
13. Observe color

Measure Absorbance using Spectrophotomer

14. Pellet the cells
15. Measure absorbance of supernatant.The supernatant was tested for absorbance using spectroscopy on a TECAN set to measure the wavelength range 350nm-750nm at 10nm intervals.
Silverbinding.jpg
Control.JPG Construct11.JPG Construct12.JPG Construct13.JPG Construct14.JPG Construct15.JPG Construct16.JPG Construct17.JPG


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1249
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1023
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1005


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Categories
Parameters
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