Team:Warsaw/Calendar-Main/5 August 2008

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Latest revision as of 09:01, 29 October 2008


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Cloning of truncated fragment of protein A (ΔA)

Emilia

Isolation of plasmids from cultures inoculated on previous day.
Control digest with SacI and BamHI (BamHI buffer).
Gel electrophoresis - proper clones found for both products of ligation.

Checking if OmpA_omega_ΔA_alpha gives ampicillin resistance

Piotr, Weronika

Inoculation OmpA_omega_ΔA_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):
ampicillin concentration (μg/mL):	0	0.1	0.25	0.5	0.75	1
25	1.558	1.469	1.587	1.49	1.566	1.311
50	1.425	1.435	1.524	1.055	0.920	0.935
75	1.09	0.989	1.447	0.971	0.951	0.992
100	0.09	0.685	1.378	1.078	0.977	0.992

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

Checking OmpA_omega_ΔA_alpha expression

Piotr, Weronika

Spinning.
Suspending.
Adding of lysis buffer.
Boiling.
Putting into poliacrylamide gel.
Transfer onto nitrocellulose.
Blocking.
Anti-A antibody binding.
Washing.
Anti-rabbit antibody binding.
Developing with BCIP and NBT (Fig. 1.).

Fig. 1.Protein A detection in bacterial lysates. 1 - protein marker, 2 and 3 - E coli without plasmid, 4 - Omp_omega_deltaA_alpha + 0,5 mM IPTG, 5 - E coli without plasmid, 6 - Omp_omega_deltaA_alpha + 1 mM IPTG, 7 - Omp_omega_A_deltaalpha without inducer.

Preparing pACYC177+OmpA_omega_ΔA construct

Michał K.

Transformation of E. coli TOP10 strain with ligation.
Transformants plating on LB + kanamycin.

@@ Line 5: / Line 5: @@
 <html>
-<h3>Cloning of truncated fragment of protein A</h3>
+<h3>Cloning of truncated fragment of protein A (&Delta;A)</h3>
-<h4>Piotr</h4>
+<h4>Emilia</h4>
 <ol><li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation</a> of plasmids from cultures inoculated on previous day.</li>
 <li> Control <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> with SacI and BamHI (BamHI buffer).</li>
-<li> Gel electrophoresis - proper clones founded for both products of ligation.</li></ol>
+<li> Gel electrophoresis - proper clones found for both products of ligation.</li></ol>
-<h3>Checking if OmpA_omega_A_alpha gives ampicillin resistance</h3>
+<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>Piotr</h4>
+<h4>Piotr, Weronika</h4>
-<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>
+<p>Inoculation <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_&Delta;A_alpha</a> 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>
 <center><h4 style="text-align: center">Measurement of bacterial culture growth (OD) in the evening:</h4> </center>
@@ Line 30: / Line 30: @@
 </table>
 <p>
-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>
+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 mmol/mL (repetition is necessary)</p>
-<h3>Checking OmpA_omega_A_alpha and OmpA_A_alpha expression</h3>
+<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>
+<h4>Piotr, Weronika</h4>
@@ Line 47: / Line 47: @@
 <li>Washing.</li>
 <li>Anti-rabbit antibody binding.</li>
-<li>Developing with BCIP and NBT.</li>
+<li>Developing with BCIP and NBT (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/5_August_2008#fig1">Fig. 1.</a>). </li>
 </ol>
-[a photo of the gel is top be put here]
+<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/e/ea/Western1_WAW.jpg" width=400/></a>
+<var><b>Fig. 1.Protein A detection in bacterial lysates.</b><br>
+- protein marker,<br>
+and 3 - <i>E coli</i> without plasmid,<br>
+- <b>Omp_omega_deltaA_alpha</b> + 0,5 mM IPTG,<br>
+- <i>E coli</i> without plasmid,<br>
+- <b>Omp_omega_deltaA_alpha</b> + 1 mM IPTG,<br>
+- Omp_omega_A_deltaalpha without inducer.</var>
+<br><br>
-<h3>Preparing pACYC177+OmpA_omega_deltaA construct</h3><h4>Michał K.</h4>
+<h3>Preparing <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-deltaA>pACYC177+OmpA_omega_&Delta;A</a> construct</h3><h4>Michał K.</h4>
 <ol><li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#electrotransform">Transformation</a> of E. coli <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> strain with ligation. </li>
 <li>Transformants plating on LB + kanamycin. </li></ol>