Team:Warsaw/Calendar-Main/5 August 2008

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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>  
<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>  
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<h4>Piotr</h4>
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<h4>Piotr, Weronika</h4>
<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>
<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>
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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>
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>
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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> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-alpha>OmpA_A_alpha</a> expression</h3>  
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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>  
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<h4>Piotr</h4>
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<h4>Piotr, Weronika</h4>
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1 - protein marker,<br>
1 - protein marker,<br>
2 and 3 - <i>E coli</i> without plasmid,<br>
2 and 3 - <i>E coli</i> without plasmid,<br>
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4 - Omp_omega_A_alpha + 0,5 mM IPTG,<br>
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4 - <b>Omp_omega_deltaA_alpha</b> + 0,5 mM IPTG,<br>
5 - <i>E coli</i> without plasmid,<br>
5 - <i>E coli</i> without plasmid,<br>
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6 - Omp_omega_A_alpha + 0,5 mM IPTG,<br>
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6 - <b>Omp_omega_deltaA_alpha</b> + 1 mM IPTG,<br>
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7 - Omp_omega_A_alpha without inducer.</var>
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7 - Omp_omega_A_deltaalpha without inducer.</var>
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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

  1. Isolation of plasmids from cultures inoculated on previous day.
  2. Control digest with SacI and BamHI (BamHI buffer).
  3. 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):
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_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

  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 (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.

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


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