Team:Warsaw/Calendar-Main/18 September 2008

'Hunter and prey' system tests: Competition tests Weronika Inoculation of pACYC177+OmpA_A_alpha, pACYC177+OmpA_Z_omega, pACYC177+OmpA_A_omega and pACYC177+OmpA_Z_alpha + induction using 0.25mM iPTG

MutD5 testing Piotr Inoculation of mutD5 into 100 ml of LB and rendering them chemocompetent. Electroporation with pACYC177+OmpA_Z_alpha, pACYC177+OmpA_Z_omega, pACYC177+OmpA_A_omega and pACYC177+OmpA_omega_&Delta;A.

Optimisation of primers for preparation of parts Michał K. <ol><li> Repetition of PCR</a> on pACYC177+OmpA_omega_&Delta;A_alpha</a> plasmid using

LinLSXNE</a> and AlphaPSpe</a> primers (elongation length 60s) to obtain linker_alpha (BBa_K103009)</a> fragment. Reaction was carried out with 25 cycles and in temperature gradient from 55 to 75 &deg;C (four reactions).</li>

<li> Gel electrophoresis of PCR products (Fig. 1.</a>).</li></ol>

<img src="http://2008.igem.org/wiki/images/c/c3/Grad2_17_09.jpg" width=180/></a> Fig. 1. Gradient PCR (temperatures:55-75 &deg;C) 1. Marker 2. 55 &deg;C linker_alpha 3. 60 &deg;C linker_alpha 4. 65 &deg;C linker_alpha 5. 70 &deg;C linker_alpha

Preparation of &Delta;A (BBa_K103003)</a> Michał K. <ol> <li>PCR</a> on pACYC177+OmpA_omega_&Delta;A_alpha</a> plasmid using

AL_BNXNE</a> and APSacSpe</a>

primers (annealing temperature 58 &deg;C; elongation length 45s) to obtain &Delta;A fragment. </li> <li> Gel electrophoresis of PCR product and gel-out</a> of proper bands (&Delta;A - 250 bp). Fig. 2</a>.</li> <li>Digest</a> of purified PCR product and pSB1A3</a> standard plasmid with EcoRI and BcuI (BamHI buffer). Dephosphorylation</a> (CIAP) of plasmid.</li> <li><a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digested PCR product.</li>

<li> Gel electrophoresis of digested plasmid and <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band (<a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> - 2200 bp).<a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/18_September_2008#fig3">Fig. 3</a>.</li></ol>

<img src="http://2008.igem.org/wiki/images/d/d0/Go_17_09.jpg" width="300"/> Fig. 2. PCR to obtain inserts 1. Marker 2. DeltaA 3. Omega 4. OmpA_omega <img src="http://2008.igem.org/wiki/images/9/96/Go2_19_09.jpg" width=180/></a> Fig. 3. EcoRI/BcuI digest of pSB1A3 1. Marker 2. Digested pSB1A3

Preparation of <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a> Michał K. <ol> <li> <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> plasmid using

<a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#LinLSXNE">LinLSXNE</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaPSpe">AlphaPSpe</a> primers (annealing temperature 65 &deg;C; elongation length 60s) to obtain <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103009>linker_alpha (BBa_K103009) fragment. </li>

<li> Gel electrophoresis of PCR product and <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands (linker_alpha - 600 bp).<a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/18_September_2008#fig4">Fig. 4</a>.</li> <li><a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of purified PCR product with EcoRI and BcuI (BamHI buffer). </li> <li><a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digested PCR product.</li> </ol>

<img src="http://2008.igem.org/wiki/images/8/85/Go_1909_2008.jpg" width=180/></a> Fig. 4. PCR to obtain linker_alpha for reisolation from agarose gel 1. Marker 2. linker_alpha

Preparation of <a href=http://partsregistry.org/Part:BBa_K103013>linker_omega (BBa_K103013)</a> Michał K. <ol> <li><a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href="http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega">pACYC177 + OmpA_omega</a> plasmid using <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#LinLSXNE">LinLSXNE</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#OmegPSpe">OmegPSpe</a> primers (annealing temperature 55 &deg;C; elongation length 60s) to obtain <a href=http://partsregistry.org/Part:BBa_K103013>linker_omega (BBa_K103013)</a> fragment. </li>

<li> Gel electrophoresis of PCR products and <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band (linker_omega - 350 bp). <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/18_September_2008#fig2">Fig. 2</a>.</li> <li><a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of purified PCR product with EcoRI and BcuI (BamHI buffer).</li>

<li><a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digested PCR product.</li>

<img src="http://2008.igem.org/wiki/images/d/d0/Go_17_09.jpg" width="300"/> Fig. 2. PCR to obtain inserts 1. Marker 2. DeltaA 3. Omega 4. OmpA_omega

</ol>

Preparation of <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a> Michał K.

<ol> <li><a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> plasmid using <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#OmpLNXNE">OmpLNXNE</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a> primers (annealing temperature 58 &deg;C; elongation length 90s) to obtain <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a> fragment.</li>

<li> Gel electrophoresis of PCR products and <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands (OmpA-linker-omega-linker - 900 bp). <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/18_September_2008#fig2">Fig. 2</a>.</li>

<li><a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of purified PCR product with EcoRI and BcuI (BamHI buffer).</li>

<li><a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digested PCR products.</li></ol> Mentioned primers were used only for test purposes. During part preparation we've used another primers.

<img src="http://2008.igem.org/wiki/images/d/d0/Go_17_09.jpg" width=220/></a> Fig. 2. PCR products for reisolation from agarose gel 1. Marker 2. deltaA 3. omega 4. ompA_omega

Preparation of <a href=http://partsregistry.org/Part:BBa_K103006>OmpA-linker (BBa_K103006)</a> Michał K.

<ol>

<li><a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-alpha>pET15b+OmpA_alpha</a> plasmid with NdeI and SacI (BamHI buffer). <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">Dephosphorylation</a> (CIAP) of plasmid.</li>

<li> Gel electrophoresis of digested plasmid and <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band (OmpA_linker - 500 bp). <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/18_September_2008#fig5">Fig. 5</a>.</li>

<img src="http://2008.igem.org/wiki/images/6/6a/Go_29_09.jpg" width=180/></a> Fig. 5. SacI/NdeI digest of pET15b_OmpA_omega 1. Marker 2. digested pSB1A3