Team:Warsaw/Calendar-Main/29 July 2008

From 2008.igem.org

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<h3>Cloning of omega_A DNA fragment to pACYC177+OmpA_alpha</h3><h4>Michał K.</h4>
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<ol><li>DNA fragment of omega_A isolated from gel on 20 July <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digestion</a> with SacI and NotI.</li>
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<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>clean-up</a>Clean-up of digested DNA fragment.</li>
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<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digestion</a> (SacI and NotI) and dephosphorylation (CIAP) of pACYC177+OmpA_alpha. </li>
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<li>Gel electrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of 4300 bp band. </li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of DNA fragments from 2. and 4. (1 hr)</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of E. coli <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> strain with ligation.</li>
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<li>Transformants plating on LB + kanamycin.</li></ol>
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Revision as of 21:16, 11 October 2008

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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).

Cloning of omega_A DNA fragment to pACYC177+OmpA_alpha

Michał K.

  1. DNA fragment of omega_A isolated from gel on 20 July digestion with SacI and NotI.
  2. clean-upClean-up of digested DNA fragment.
  3. Digestion (SacI and NotI) and dephosphorylation (CIAP) of pACYC177+OmpA_alpha.
  4. Gel electrophoresis and gel-out of 4300 bp band.
  5. Ligation of DNA fragments from 2. and 4. (1 hr)
  6. Transformation of E. coli TOP10 strain with ligation.
  7. Transformants plating on LB + kanamycin.

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.