Overview
To make contribution for future iGEM teams, we did some useful work. First, we added some information learned from literatures to an existing part K4167660 (Part: BBa K4167660 - parts.igem.org), as well as new data collected from our experiments. In addition, we created a new part (Part: BBa K4677001 - parts.igem.org). We have documented them on the Part’s Main Page on the Registry. Also, they are shown as follows:
1. Improvement of an existing part K4167660
1.1 New documentation to K4167660
K4167660 is a LacZ (β-galactosidase) generator driven by Plac. So, we added some documentation related to β-galactosidase. β-galactosidase has three enzymatic activities (Fig. 1). First, it can cleave the disaccharide lactose to form glucose and galactose, which can then enter glycolysis. Second, the enzyme can catalyze the transgalactosylation of lactose to allolactose, and, third, the allolactose can be cleaved to the monosaccharides. It is allolactose that binds to lacZ repressor and creates the positive feedback loop that regulates the amount of β-galactosidase in the cell [1].
Fig.1 Schematic summarizing the roles of β-galactosidase in the cell.
The enzyme can hydrolyze lactose to galactose plus glucose, it can transgalactosylate to form allolactose, and it can hydrolyze allolactose. The presence of lactose results in the synthesis of allolactose which binds to the lac repressor and reduces its affinity for the lac operon. This in turn allows the synthesis of β-galactosidase, the product of the lacZ gene.
β-galactosidase is a tetramer of four identical polypeptide chains, each of 1023 amino acids. Within each monomer, the 1023 amino acids form five well-defined structural domains. The third (central) domain (residues 334–627) is a so-called triose phosphate isomerase (TIM) or α8β8 barrel with the active site forming a deep pit at the C-terminal end of this barrel (Fig.2). As noted below, critical elements of the active site are also contributed by amino acids from elsewhere in the same polypeptide chain as well as from other chains within the tetramer [2].
Fig.2 The backbone structure of β-galactosidase tetramer.
Domain 1, blue; Domain 2, green; Domain 3, yellow; Domain 4, cyan; Domain 5, red. Lighter and darker shading is used to differentiate equivalent domains in different subunits. Metal ions are shown as spheres, Na+, green; Mg++, blue.
The crystal structure of β-galactosidase was determined in an orthorhombic crystal with a single tetramer in the asymmetric unit (Fig.3) [2].
Fig.3 Demonstration that β-galactosidase in crystals is catalytically active.
Crystal of β-galactosidase (orthorhombic; ca.0.2 mm) in the absence (left) and in the presence, after about 2 h, of the substrate X-gal (right).
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1.2 New data from our lab to K4167660
Since the part K4167660 is a β-galactosidase generator which can catalyze the substrate X-gal to generate blue product. To detect the part whether could express the functional enzyme or not, we cultured the BL21 cells transformed with the part K4167660 on the agar plate in the presence of IPTG (0.5 mM) and X-gal (40 µg/mL). Some blue clones were observed (Fig.4).
Fig.4 The blue clones were observed on the agar plate after the part K4167660 was transformed into BL21 cells in the presence of IPTG and X-gal.
A: Negative control without IPTG; B: experiment group with IPTG (0.5 mM) and X-gal (40 µg/mL).
References
[1] Juers DH, Matthews BW, Huber RE. LacZ β-galactosidase: structure and function of an enzyme of historical and molecular biological importance. Protein Sci. 2012; 21(12):1792-807.
[2] Juers DH, Wigley RH, Zhang X, Huber RE, Tronrud DE, Matthews BW. High resolution refinement of β-galactosidase in a new crystal form reveals multiple metal binding sites and provides a structural basis for a-complementation. Protein Sci. 2000, 9:1685–1699.
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2. Creation of a new part K4677001
2.1 Introduction to part K4677001
K4677001 is a basic part containing TorR gene (693 bp) of E. coli. It encodes about 25 kDa response regulator protein involved in the expression regulation of the trimethylamine N-oxide (TMAO) reductase genes, which are controlled by TorCAD operon. The TorR gene is located just upstream of the TorCAD operon, with an opposite transcription direction. TheTorR-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 expression of TMAO reductase dependently on TMAO.
2.2 Construction and identification
In order to construct the standard part pSB1C3-TorR plasmid, TorR sequence was tested to see if there is EcoR I and Pst I site. The testing result was shown in Fig.5.
Fig.5 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.6):
Fig.6 Identification of standard part pSB1C3-TorR using digestion with EcoR I and Pst I, and PCR method.
M: Marker; 1: Plasmid; 2: Digestion result; 3: PCR result.
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2.3 Expression and purification
In our project, TorR was expressed in pET28a 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.7):
Fig.7 The SDS-PAGE result shows expression and purification of TorR protein.
M: Marker; 1: All supernatant proteins containing overexpression TorR induced by IPTG; 2: Supernatant proteins not bound to magnetic beads in purification process; 3: Proteins in wash buffer; 4: Purified TorR (27 kDa) protein in elution buffer. .
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2.4 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.