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Team:Warsaw/Calendar-Main/11 August 2008
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/7/77/Western2_WAW.jpg" width=400/></a> | <a name="fig1"><img src="https://static.igem.org/mediawiki/2008/7/77/Western2_WAW.jpg" width=400/></a> | ||
| - | <var>Detection of protein A. | + | <var><b>Detection of protein A in bacterial lysates.</b><br> |
| + | 1 - protein marker,<br> | ||
| + | 2 - <i>E. coli</i> without plasmid,<br> | ||
| + | 3, 4, 5, 6 - <b>Omp_omega_A_alpha fusion protein:</b><br> | ||
| + | 3 - in Top10 strain without inducer,<br> | ||
| + | 4 - in Top10 strain with IPTG,<br> | ||
| + | 5 - in Rosetta strain without inducer,<br> | ||
| + | 6 - in Rosetta strain with IPTG,<br> | ||
| + | 7, 8, 9, 10 - <b>Omp_A_alpha fusion protein:</b><br> | ||
| + | 7 - in Top10 strain without inducer,<br> | ||
| + | 8 - in Rosetta strain without inducer,<br> | ||
| + | 9 - in Top10 strain with IPTG,<br> | ||
| + | 10 - in Rosetta strain with IPTG.</var> | ||
<br> | <br> | ||
Revision as of 22:38, 26 October 2008
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Checking whether degradation of the fusions with OmpA is caused by Lon and OmpT proteases (present in TOP10)PiotrTest was conducted in E.coli Rosetta strain expressing OmpA_omega_ΔA_alpha (with and without induction) and OmpA_ΔA_alpha.
Detection of protein A in bacterial lysates.1 - protein marker, 2 - E. coli without plasmid, 3, 4, 5, 6 - Omp_omega_A_alpha fusion protein: 3 - in Top10 strain without inducer, 4 - in Top10 strain with IPTG, 5 - in Rosetta strain without inducer, 6 - in Rosetta strain with IPTG, 7, 8, 9, 10 - Omp_A_alpha fusion protein: 7 - in Top10 strain without inducer, 8 - in Rosetta strain without inducer, 9 - in Top10 strain with IPTG, 10 - in Rosetta strain with IPTG. We didn't observe differences in expression and degradation in Rosettas nor in TOP10. Therefore we suppose that degradation of the fusions is caused by factor other than Lon and OmpT proteases. Purification of proteins: Z-alpha and Z-omegaEmilia100 ml of medium inoculated with overnight culture, then used as inoculum for 1 l culture in presence of ampicillin, kanamycin and IPTG (according to protocol).
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