Difference between revisions of "Part:BBa K1758301"
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| − | This part consists of the arsenic repressor (arsR) of the chromosomal arsenic operon of <i>E. coli</i> under control of the T7 promoter. The gene includes a C-terminal his-tag and can therefore be used for purification of | + | This part consists of the arsenic repressor (<i>arsR</i>) of the chromosomal arsenic operon of <i>E. coli</i> under control of the T7 promoter. The gene includes a C-terminal his-tag and can therefore be used for purification of ArsR. |
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<img src="https://static.igem.org/mediawiki/parts/1/10/Bielefeld-CeBiTec_2015_SDS_PAGE_ArsR.png"> | <img src="https://static.igem.org/mediawiki/parts/1/10/Bielefeld-CeBiTec_2015_SDS_PAGE_ArsR.png"> | ||
| − | <br> | + | <br><br> |
| + | <h2>Test in cell-free protein synthesis</h2> | ||
<p>The eluted protein was added to a cell-free protein synthesis reaction together with <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1758300">BBa_K1758300</a>, which is an sfGFP gene under control of the T7 promoter and the arsenic operator. As opposed to our expectation, we observed no repression in the presence of ArsR. We assume that the ArsR concentration was too low to observe a repression. Furthermore, the position of the arsenic operator in <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1758300">BBa_K1758300</a> is not ideal for an effective repression.</p> | <p>The eluted protein was added to a cell-free protein synthesis reaction together with <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1758300">BBa_K1758300</a>, which is an sfGFP gene under control of the T7 promoter and the arsenic operator. As opposed to our expectation, we observed no repression in the presence of ArsR. We assume that the ArsR concentration was too low to observe a repression. Furthermore, the position of the arsenic operator in <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1758300">BBa_K1758300</a> is not ideal for an effective repression.</p> | ||
Latest revision as of 05:28, 19 September 2015
His-tagged arsenic repressor (ArsR) under control of the T7 promoter
This part consists of the arsenic repressor (arsR) of the chromosomal arsenic operon of E. coli under control of the T7 promoter. The gene includes a C-terminal his-tag and can therefore be used for purification of ArsR.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 177
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Expression and purification
We expressed this protein in E. coli KRX and purified it under native conditions with the Protino® Ni-TED 1000 Packed Columns Kit according to the manufacturer´s protocol. We took samples of the purification steps and ran them on an SDS PAGE to check if the purification was successful. ArsR is a dimer with 26.6 kDa. In all three elution fractions there is a band which corresponds to this size, so we assume that the expression and purification was successful. However, the elution fractions include several other proteins. The lowest band might correspond to the monomer, the other proteins are probably the result of unspecific binding to the Ni-TED column.
Test in cell-free protein synthesis
The eluted protein was added to a cell-free protein synthesis reaction together with BBa_K1758300, which is an sfGFP gene under control of the T7 promoter and the arsenic operator. As opposed to our expectation, we observed no repression in the presence of ArsR. We assume that the ArsR concentration was too low to observe a repression. Furthermore, the position of the arsenic operator in BBa_K1758300 is not ideal for an effective repression.