Team:Warsaw/Calendar-Main/4 August 2008

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<h3> Cloning of truncated fragment of protein A</h3>  
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<h3> Cloning of truncated fragment of protein A (&Delta;A)</h3>  
<h4>Piotr</h4>
<h4>Piotr</h4>
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<p>Inoculation of  some pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA colonies of tranformnts.</p>
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<p>Inoculation of  some <a href=phttps://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-deltaA-alpha>pACYC177+OmpA_alpha + &Delta;A</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BOmpA-deltaA-omega>pACYC177+OmpA_omega + &Delta;A</a> colonies of tranformants.</p>
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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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<h3>Checking <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_&Delta;A_alpha</a> expression</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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<p>Inoculation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_&Delta;A_alpha</a> with inductor (0,5 mmol/mL IPTG) and negative control without IPTG.</p>
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<h3>Checking if omp_omega_A_alpha gives ampicillin resistance<br>
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<h3>Checking if <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_&Delta;A_alpha</a> gives ampicillin resistance</h3><h4>Emilia</h4>
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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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<p>Inoculation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_&Delta;A_alpha</a> into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75, 1 mmol/mL.</p>
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<h3>Preparing pACYC177+OmpA_omega_&Delta;A construct</h3><h4>Michał K.</h4>
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<ol><li><a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#digest">Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> (from 25 July) with BamHI and NotI (BamHI buffer). DNA ends <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#blunting">blunting</a> with Klenow fragment (3 hr). </li>
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<li>Gel elctrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/4_August_2008#fig1">Fig. 1</a>) and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band - 4300 bp. </li>
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<li>Overnight <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#ligation">ligation</a> of isolated DNA fragment. </li>
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</ol>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/1/17/Gucio.jpg" width=240/></a> <var><b>Fig. 1. </b>Gel-out of pACYC177_OmpA_omega_deltaA_alpha digested with NotI/BamHI after blunting ends.<br>
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1. Marker<br>
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2. pACYC177_OmpA_omega_deltaA_alpha <br></var>
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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>
 
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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). 
 
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</p>
 
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<h3>Checking if OmpA_omega_A_alpha gives ampicillin resistance<br>
 
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Piotr</h3>
 
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<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>
 
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<h3>Checking OmpA_omega_A_alpha and OmpA_A_alpha expression<br>
 
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Piotr</h3>
 
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<ol>
 
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<li>Spinning</li>
 
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<li>Suspending</li>
 
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<li>Adding of lysis buffer</li>
 
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<li>Boiling</li>
 
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<li>Putting into poliacrylamide gel</li>
 
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<li>Transfer onto nitrocellulose</li>
 
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<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>
 
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<h3>Checking for presence of A on the cell membrane</h3>
 
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<h4>Piotr</h4>
 
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<p>Inoculation of omp_A_alfa, omp_Z_alfa and omp_omega_A_alfa (with and without induction).</p>
 

Latest revision as of 10:50, 29 October 2008

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Cloning of truncated fragment of protein A (ΔA)

Piotr

Inoculation of some pACYC177+OmpA_alpha + ΔA and pACYC177+OmpA_omega + ΔA colonies of tranformants.

Checking OmpA_omega_ΔA_alpha expression

Piotr

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

Checking if OmpA_omega_ΔA_alpha gives ampicillin resistance

Emilia

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

Preparing pACYC177+OmpA_omega_ΔA construct

Michał K.

  1. Digest of pACYC177+OmpA_omega_ΔA_alpha (from 25 July) with BamHI and NotI (BamHI buffer). DNA ends blunting with Klenow fragment (3 hr).
  2. Gel elctrophoresis (Fig. 1) and gel-out of proper band - 4300 bp.
  3. Overnight ligation of isolated DNA fragment.
Fig. 1. Gel-out of pACYC177_OmpA_omega_deltaA_alpha digested with NotI/BamHI after blunting ends.
1. Marker
2. pACYC177_OmpA_omega_deltaA_alpha