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| | + | <h3> Cloning of truncated fragment of protein A (ΔA)</h3> |
| | + | <h4>Piotr</h4> |
| | + | <p>Inoculation of some <a href=phttps://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-deltaA-alpha>pACYC177+OmpA_alpha + ΔA</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BOmpA-deltaA-omega>pACYC177+OmpA_omega + ΔA</a> colonies of tranformants.</p> |
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| | + | <h3>Checking <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_ΔA_alpha</a> expression</h3><h4>Piotr</h4> |
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| - | <h3>Checking the expression of omp_omega_A_alpha and omp_A_alpha</h3><h4>Piotr</h4> | + | <p>Inoculation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_ΔA_alpha</a> with inductor (0,5 mmol/mL IPTG) and negative control without IPTG.</p> |
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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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| | + | <h3>Checking if <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_ΔA_alpha</a> gives ampicillin resistance</h3><h4>Emilia</h4> |
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| - | <h3>Checking if omp_omega_A_alpha gives ampicillin resistance<br> | + | <p>Inoculation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_ΔA_alpha</a> into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75, 1 mmol/mL.</p> |
| - | 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> | + | <h3>Preparing pACYC177+OmpA_omega_ΔA construct</h3><h4>Michał K.</h4> |
| | + | <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_ΔA_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> |
| | + | <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> |
| | + | <li>Overnight <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#ligation">ligation</a> of isolated DNA fragment. </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°C to 75°C)<br> | + | <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> |
| - | - Optimization of number of cycles(15, 20, 25, 30, 35)<br>
| + | 1. Marker<br> |
| | + | 2. pACYC177_OmpA_omega_deltaA_alpha <br></var> |
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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°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>
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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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| - | <h4>Michał K.</h4>
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| - | <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>
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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>
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"
Fig. 1. Gel-out of pACYC177_OmpA_omega_deltaA_alpha digested with NotI/BamHI after blunting ends.
