Part:BBa_K4677001
TorR
The torR gene of Escherichia coli encodes a 25kD response regulator protein involved in the expression regulation of the trimethylamine N-oxide reductase genes, which are regulated by torCAD operon. The torR gene is located just upstream of the torCAD operon, with an opposite transcription direction. The torR-torCAD intergenic region is unusual in that it contains four direct repeats of a 10-nucleotide motif. Part or all of these motifs could be involved in the binding of TorR protein. TorR only mediate the TMAO induction.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
In order to construct the standard part pSB1C3-TorR plasmid, TorR sequence was tested to see if there is EcoRI and PstI site. The testing result was shown in Fig.1.
Fig.1 The map of TorR gene sequence described by SnapGene Viewer, showing the restriction enzyme information (no EcoRI and PstI sites).
After testing the restriction enzyme information of TorR gene using SnapGene software, it was inserted into the pSB1C3 plasmid to construct the standard part pSB1C3-TorR with PCR method. Then it was identified as follows:
Fig.2 Identification of standard part pSB1C3-TorR using digestion with EcoRI and PstI, and PCR method.
M: Marker; 1: Plasmid; 2: Digestion result; 3: PCR result.
In our project, TorR was expressed in pET-28a vector and purified by 6×his tag, After induced expression by IPTG, TorR protein was purified using magnetic beads. The SDS-PAGE electrophoresis result was shown as follows:
Fig.3 The SDS-PAGE result shows expression and purification of TorR protein.
M: Marker; 1: All supernatant proteins containing overexpressioned TorR induced by IPTG; 2: Supernatant proteins not bound to magnetic beads in purification process; 3: Proteins in wash buffer; 4: Purified TorR (27kD) protein in elution buffer.
Summary:
The part K4677001 was constructed successfully. TorR gene could be expressed on the pET-28a expressing vector in the presence of IPTG induction. This part could be used for studying the TMAO reductase pathway in which TorR involved.
Refferences
[1]Moore JO, Hendrickson WA. An asymmetry-to-symmetry switch in signal transmission by the histidine kinase receptor for TMAO. Structure. 2012; 20(4): 729-741.doi:10.1016/j.str.2012.02.021
[2]Tang WH, Wang Z, Levison BS, Koeth RA, Britt EB, Fu X, et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med 2013; 368(17):1575-1584.
[3]Senthong V, Wang Z, Fan Y, Wu Y, Hazen SL, Tang WH. Trimethylamine N-Oxide and Mortality Risk in Patients With Peripheral Artery Disease. J Am Heart Assoc 2016; 5(10).
| None |