Team:Warsaw/Calendar-Main/4 August 2008

From 2008.igem.org

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<html>
<html>
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<h3>Checking the expression of omp_omega_A_alpha and omp_A_alpha</h3><h4>Piotr</h4>
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<p>Inoculation of omp_omega_A_alpha and omp_A_alpha with inductor (0,5 mmol/mL IPTG) and negative control without IPTG.</p>
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 +
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<h3>Checking if omp_omega_A_alpha gives ampicillin resistance<br>
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Piotr</h3>
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<ol><li>Inoculation of omp_omega_A_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75, 1 mmol/mL</li></ol>
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<center><h4><p>1. Optimization of PCR to obtain truncated fragment of protein A DNA </center>
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</h4>
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Primers: <html>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI ">AL+SacI</a>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> </html>
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Elongation time: 30s <br>
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 +
- Optimization of annealing temperature (gradient from 55&deg;C to 75&deg;C)<br>
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- Optimization of number of cycles(15, 20, 25, 30, 35)<br>
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 +
2. PCR to obtain truncated A protein DNA fragment <br>
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<p>Primers:<html>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI ">AL+SacI</a>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a></html></p>
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Elongation time: 30s <br>
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 +
Annealing temperature: 60&deg;C <br>
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20 cycles <br>
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3. Gel electrophoresis and isolation of 250 bp band. <br>
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4. Digest of isolated PCR product, pACYC177+OmpA_alpha and pACYC177+OmpA_omega with NotI and SacI. <br>
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5. Clean-up of digestion reaction. <br>
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6. Gel electrophoresis for estimation of DNA concentration. <br>
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7. Overnight ligation: pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA.
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<html>
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<center><h4>Checking if degradation of fusion with OmpA is a result of Top10 proteases' activity (lon, iompt)</center>Piotr:</h4>
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<br>
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Inoculation of OmpA_omega_A_alpha and OmpA_A_alpha with and without inductor (0,5 mm/mL IPTG) in <i>E. coli</i> Rosetta strain.
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<p>1. Isolation of plasmids from cultures inocluated on previous day.<br>
 +
2. Control digest of isolated plasmids with BamHI and SacI (we confirmed 4 colonies but A in our constructs turned out to be trunctated and contain only one from two highly similar domains). 
 +
</p>
 +
 +
<h3>Checking if OmpA_omega_A_alpha gives ampicillin resistance<br>
 +
Piotr</h3>
 +
 +
<p>Inoculation OmpA_omega_A_alpha from various IPTG concentrations (in mmol/mL) (0, 0.1, 0.25, 0.5, 0.75, 1) into same IPTG concentrations, but with various ampicillin concentrations (in mmol/mL)(25, 50, 75, 100) in ratio: 1:50.</p>
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<center><h4 style="text-align: center">Measurement of bacterial culture growth (OD) in the evening:</h4> </center>
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<table id="result" align="center">
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<tr><th rowspan="2">ampicillin concentration (μg/mL):</th><th colspan="6">IPTG concentration (mmol/mL):</td></tr>
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<tr><th>0</th><th>0.1</th><th>0.25</th><th>0.5</th><th>0.75</th><th>1</th></tr>
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<tr><th>25</th><td class="live">1.558</td><td class="live">1.469</td><td class="live">1.587</td><td class="live">1.49</td><td class="live">1.566</td><td class="live">1.311</td></tr>
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<tr><th>50</th><td class="live">1.425</td><td class="live">1.435</td><td class="live">1.524</td><td class="live">1.055</td><td class="live">0.920</td><td class="live">0.935</td></tr>
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<tr><th>75</th><td class="live">1.09</td><td class="live">0.989</td><td class="live">1.447</td><td class="live">0.971</td><td class="live">0.951</td><td class="live">0.992</td></tr>
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<tr><th>100</th><td class="live">0.09</td><td class="live">0.685</td><td class="live">1.378</td><td class="live">1.078</td><td class="live">0.977</td><td class="live">0.992</td></tr>
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</table>
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<p>
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Inoculation of OmpA_omega_A_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75 mmol/mL (replication is necessary)</p>
 +
 +
<h3>Checking OmpA_omega_A_alpha and OmpA_A_alpha expression<br>
 +
Piotr</h3>
 +
 +
 +
<ol>
 +
<li>Spinning</li>
 +
<li>Suspending</li>
 +
<li>Adding of lysis buffer</li>
 +
<li>Boiling</li>
 +
<li>Putting into poliacrylamide gel</li>
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<li>Transfer onto nitrocellulose</li>
 +
<li>Blocking</li>
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<li>Anti-A antibody binding</li>
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<li>Washing</li>
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<li>Anti-rabbit antibody binding</li>
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<li>Developing with BCIP and NBT</li>
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</ol>
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[a photo of the gel is top be put here]
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<h4>Michał L., Ewa, Marcin</h4>
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<p>
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<ol>
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<li>Separate transformant colonies (transformation from previous day) inoculated to liquid LB with kanamycin. </li>
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<li>Inoculation of pACYC177+OmpA_alpha and pACYC177+OmpA_omega - liquid LB with kanamycin.</li></ol>
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</p>
<h4>Michał K.</h4>
<h4>Michał K.</h4>
<p>Separate transformant colonies (tranformations from <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/3_August_2008">previous day</a>) inoculated to liquid LB with kanamycin</p>
<p>Separate transformant colonies (tranformations from <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/3_August_2008">previous day</a>) inoculated to liquid LB with kanamycin</p>

Revision as of 21:54, 11 October 2008

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Checking the expression of omp_omega_A_alpha and omp_A_alpha

Piotr

Inoculation of omp_omega_A_alpha and omp_A_alpha with inductor (0,5 mmol/mL IPTG) and negative control without IPTG.

Checking if omp_omega_A_alpha gives ampicillin resistance
Piotr

  1. Inoculation of omp_omega_A_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75, 1 mmol/mL

1. Optimization of PCR to obtain truncated fragment of protein A DNA

Primers: AL+SacI AP+NotI

Elongation time: 30s

- Optimization of annealing temperature (gradient from 55°C to 75°C)
- Optimization of number of cycles(15, 20, 25, 30, 35)

2. PCR to obtain truncated A protein DNA fragment

Primers: AL+SacI AP+NotI

Elongation time: 30s

Annealing temperature: 60°C

20 cycles

3. Gel electrophoresis and isolation of 250 bp band.
4. Digest of isolated PCR product, pACYC177+OmpA_alpha and pACYC177+OmpA_omega with NotI and SacI.
5. Clean-up of digestion reaction.
6. Gel electrophoresis for estimation of DNA concentration.
7. Overnight ligation: pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA.


Checking if degradation of fusion with OmpA is a result of Top10 proteases' activity (lon, iompt)

Piotr:
Inoculation of OmpA_omega_A_alpha and OmpA_A_alpha with and without inductor (0,5 mm/mL IPTG) in E. coli Rosetta strain.

1. Isolation of plasmids from cultures inocluated on previous day.
2. Control digest of isolated plasmids with BamHI and SacI (we confirmed 4 colonies but A in our constructs turned out to be trunctated and contain only one from two highly similar domains).

Checking if OmpA_omega_A_alpha gives ampicillin resistance
Piotr

Inoculation OmpA_omega_A_alpha from various IPTG concentrations (in mmol/mL) (0, 0.1, 0.25, 0.5, 0.75, 1) into same IPTG concentrations, but with various ampicillin concentrations (in mmol/mL)(25, 50, 75, 100) in ratio: 1:50.

Measurement of bacterial culture growth (OD) in the evening:

ampicillin concentration (μg/mL):IPTG concentration (mmol/mL):
00.10.250.50.751
251.5581.4691.5871.491.5661.311
501.4251.4351.5241.0550.9200.935
751.090.9891.4470.9710.9510.992
1000.090.6851.3781.0780.9770.992

Inoculation of OmpA_omega_A_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75 mmol/mL (replication is necessary)

Checking OmpA_omega_A_alpha and OmpA_A_alpha expression
Piotr

  1. Spinning
  2. Suspending
  3. Adding of lysis buffer
  4. Boiling
  5. Putting into poliacrylamide gel
  6. Transfer onto nitrocellulose
  7. Blocking
  8. Anti-A antibody binding
  9. Washing
  10. Anti-rabbit antibody binding
  11. Developing with BCIP and NBT
[a photo of the gel is top be put here]

Michał L., Ewa, Marcin

  1. Separate transformant colonies (transformation from previous day) inoculated to liquid LB with kanamycin.
  2. Inoculation of pACYC177+OmpA_alpha and pACYC177+OmpA_omega - liquid LB with kanamycin.

Michał K.

Separate transformant colonies (tranformations from previous day) inoculated to liquid LB with kanamycin

Checking for presence of A on the cell membrane

Piotr

Inoculation of omp_A_alfa, omp_Z_alfa and omp_omega_A_alfa (with and without induction).

Retrieved from "http://2008.igem.org/Team:Warsaw/Calendar-Main/4_August_2008"