Difference between revisions of "Part:BBa M10210"

Latest revision as of 14:52, 11 May 2009

{Pbad.rbs.prepro.StrepTag}{AG4}{CPG_L6!}{dblTerm}

Composite part with an Arabinose promoter {Pbad}, ribosome binding site {rbs}, prepro periplasmic targetting sequence {prepro}, a streptavidin-binding tag {StrepTag}, a silver binding peptide {AG4}, circularly permutated ompG {CPG_L6} which functions as the carrier protein for display of AG4 on the cell surface of E.coli, and a terminator {dblTerm}

Strepavidin-Binding Assay

Goals

1) To measure for the ability of the 16 display constructs to bind Strepavidin on the cell surface
2) To devise a method for quantifying the relative amount of Strepavidin bound by the constructs

The 16 Constructs Tested

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}

Controls

1)pBca9145-Bca9494    {AraC-Pbad}{rbs.cpx}
(positive control that displays cpx, a streptavidin binding peptide under Pbad)
2)DH10B (no plasmid, negative control)
3)pBca9495CA-Bca1144  {Ptet}{rbs1}{mRFP-3*}{b0015}
(negative control: same vector as the other constructs with a part that does not bind Streptavidin)

Procedure

Transforming and Plating

1) In a 96-well PCR plate, add to wells a mixture of 220uL competent cells, 30ul KCM salts, and 50 uL ddH2O. 2) Add 1uL of a construct to each well.
2) Incubate for 10' on ice, heat shock at 42C for 1.5', cool for another 2', and then add 90uL of LB media. Cover and shake for 15' at 37C.
3) Plate on chloramphenacol and incubate at 37C for 24h.

Inoculating

1) For each construct, pick 1 colony and inoculate in 4 mL of appropriate antibiotic media (CA in most cases), w/ or w/o arabinose (1:1000), in a 24 well block.
2) Shake at 37C for 16-20h.

Assaying Strepavidin Binding: First Try

1) Prefill wells in a clean 96-well skirted plate with 300uL PBS, and add 25uL of saturated culture of each construct.
2) Add 15uL Strepavidin-Phycoerythrin to each well and incubate at 37C without shaking for 30min to 1 hour.
3) Spin down the cells at 3,500 RPM for 5 min and note which pellets appear red in normal light and bright white under UV light (have bound streptavidin).
4) Decant and resuspend cells in 150uL, transfer to a microtiter plate, and measure transmittance at 575nm using 488nm excitation (phycoerythrin setting).

Assaying Strepavidin Binding: Second Try

1) Spin down 600uL of saturated culture at 5,500 RPM for 5 min in a 96-well skirted plate.
2) Remove media and resuspend in 300uL of PBS.
3) Add 1uL Strepavidin-Phycoerythrin to each well and incubate at 37C without shaking for 30min to 1 hour.
4) Spin down the cells at 5,500 RPM for 5 min and note which pellets appear red in normal light and bright white under UV light (have bound streptavidin).
5) Decant and resuspend cells in 150uL, transfer to a microtiter plate, and measure transmittance at 575nm using 488nm excitation (phycoerythrin setting).

Assaying Strepavidin Binding: Third Try

1) Spin down 200uL of saturated culture at 5,500 RPM for 5 min in a 96-well block.
2) Remove media and resuspend in 200uL of PBS.
3) Add 1uL Strepavidin-Phycoerythrin to each well and incubate at 37C without shaking for 30min to 1 hour.
4) Spin down the cells at 5,500 RPM for 5 min and note which pellets appear red in normal light and bright white under UV light (have bound streptavidin).
5) Transfer the supernatant to a microtiter plate to measure how much streptavidin was pulled out of solution by the cells (by measuring transmittance at 575nm using 488nm excitation (phycoerythrin setting)).

Assaying Streptavidin Binding: Fourth Try (some quantitative data obtained)

1) In a 96-well PCR plate, add 100uL of PBS and 10uL of saturated culture of each sample (four replicates of each).
2) Add concentrations of 0.5uL, 1uL, 2uL, or 5uL of Streptavidin-phycoerythrin to each replicate.
3) Incubate at 37C without shaking for 30 minutes to 1 hour.
4) Spin down the plate at 5,500rpm for 5 minutes.
5) Use the Tecan to measure fluorescence from the top of the plate (we did depths of 5,100um and 10,100um). 6) Normalize fluorescence to OD measurements of each sample. For accuracy, measure the optical density at 600nm (OD600) of a 10X dilution of the saturated culture and then calculate the actual OD600 of the culture.

Results

First Try

Visually, determined that the following four constructs bound to streptavidin (red color in the cell pellet as well as fluorescence under UV). The positive control (pBca9145-Bca9494) and the following 4 constructs bound to Streptavidin:

M10219	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<eCPX!}{dblTerm}
M10220	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm}
M10222	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm}
M10223	{Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm}

After we spun down the cells and resuspended in PBS, we had lost so many of the cells that we were unable to get a meaningful measurement using the Tecan.

Second Try

The same four constructs and positive control showed binding when examined visually. However, when we tried to quantify, the light scattering from the excessive number of cells interfered with the measurement and we got no meaningful data.

Third Try

The same four constructs and positive control showed binding when examined visually. However, we were unable to get a firm enough pellet to remove the supernatant without disturbing the pellet.

Fourth Try: Quantitative Data

We measured fluorescence of the supernatant at two different depths (5,100um and 10,100um). There were higher measurements of fluorescence at lower depths because the unbound streptavidin appears to concentrate lower in the wells after they have been spun down (the average difference in fluorescence intensity at the two depths was 367 for 0.5uL streptavidin and 638 for 1uL streptavidin). We only took measurements for samples that were incubated with 0.5uL or 1uL of streptavidin because higher amounts of streptavidin showed a clear visual gradient of the dye in solution.

The results shown below are an average of the fluorescence measurements obtained at 5,100um and 10,100um. We normalized the values to OD600 measurements of the saturated samples.

M10219	 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<eCPX!}{dblTerm}
M10220	 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm}
M10222	 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm}
M10223	 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm}
pBca9145-Bca9494    {AraC-Pbad}{rbs.cpx}              (positive control)
pBca9495CA-Bca1144  {Ptet}{rbs1}{mRFP-3*}{b0015}      (negative control)

For some undetermined reason the positive control did not work on the fourth try (we could not see red color/fluorescence even when we visually observed it and the quantitative results are comparable with the negative control). In this experimental trial, we visually saw binding for only 3 of the constructs: M10220, M10222, and M10223.

Analysis

The values that we obtained from the quantitative fluorescence measurements were somewhat unexpected. We were unable to explain why the fluorescence intensity measurements at 0.5uL and 1uL for these constructs were not significantly lower than the corresponding measurements for the other constructs. We would have expected such a result because increased binding should have resulted in decreased streptaviding (and fluorescence) in the supernatant that we measured. We feel that the measurements might be erroneous because of the bleed through of fluorescence from the pelleted cells.

We found it difficult to find any significance in the fluorescence intensity measurements unless we looked at the comparison between the values obtained at the two different arabinose concentrations. The three constructs that appeared to be binding streptavidin successfully (M10220, M10222, M10223), all had higher fluorescence intensity in the supernatant with 0.5uL streptavidin than they did with 1uL streptavidin. This seems to be because these constructs are binding to more streptavidin (and thereby, pulling more streptavidin out of the supernatant) as the amount of streptavidin increases. It was visually inconclusive whether or not M10219 had bound streptavidin and the quantitative results for it indicate that it more similar to the constructs that are not binding streptavidin (where the fluorescence intensity of the supernatant increases in the presence of more streptavidin).

The inconsistencies in the data (the unexplained lack of binding of the positive control in the fourth try) as well as the unexpectedly high fluorescence measurements obtained for the supernatant of M10220, M10222, and M10223 warrant further considerations. The best protocol for this experimented still needs to be determined because we were not able to get the expected quantitative data even after four tries. Better data can probably be obtained if we can find a way to separate the pellet and the supernatant without disturbing the pellet or using an excessively high amount of cells.

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.

Sequence and Features