Difference between revisions of "Part:BBa K902073"

Latest revision as of 16:47, 22 October 2020

mntP promoter

Encodes for the promoter in the putative efflux pump mntP. This promoter is responsible for maintaining manganese homeostasis in the cell. mntP promoter is activated when there is high levels of manganese in the cells. It works in tandem with the mntP riboswitch (BBa_K902074). Waters et al, 2011 show that the mntP promoter and riboswitch get activated by 10µM of MnCl₂.

Contribution by iGEM Team Tuebingen 2020

Group: Team Tuebingen 2020

Author: Lea Vogt, Lina Widerspick

Summary:

Usage

Inducible promoters are often used in synthetic biology, because they allow predictable regulation of gene expression. Induction can happen through exogenous molecules like metals, lactose, rhamnose, arabinose or anhydrotetracycline (1, 2) to mention a few, but there are also temperature or pH sensible promoter systems (3–6).

Our team, iGEM Tuebingen 2020, decided to use this promoter as an additional regulatory element in our manganese-biosensor. It was combined together with a manganese riboswitch (BBa_K902074) to control the expression of a fluorescence-tagged (FAST-2-Tag BBa_K3510000) phytochelatin (BBa_K1321005) or a chromoprotein (BBa_K864401). To assess the effect of this inducible promoter, we also design a construct with the constitutive Anderson-Promoter (BBa_J23102) as a control. You can find our four different constructs with the following part numbers: BBa_K3510002, BBa_K3510003, BBa_K3510004, and BBa_K3510005.

Biology

In 2011, Waters et. al. found the gen mntP, regulated by this promoter, when they investigated the transcriptional regulator MntR in E. coli [7]. This is also the paper, where Team Calgary 2012 obtained the sequence for this biobrick. The transcription factor MntR is now known to control manganese homeostasis by repressing the manganese importer MntH [8] and upregulating MntP [7], a putative manganese efflux pump. Interestingly, the authors found that the regulation of the MntP manganese exporter consists of two independently contributing elements (Figure 1). First, an inducible promoter allows for transcription, when activated by the regulatory proteins MntR and Fur. These only bind when cells are exposed to high manganese levels and antagonize the repressive effects of histone-like proteins [9]. Second, a manganese riboswitch (BBa_K902074) in the 5’ untranslated region (UTR) [9].

Manganese Level Regulation

Figure 1: Regulation of manganese levels in E. coli. During high cellular manganese levels the regulators MntR (yellow) and Fur activate the promoter (green) of the mntP gene (purple). This allows for transcription, but binding of manganese to the riboswitch (orange) is necessary for following translation and expression of the manganese efflux pump MntP (purple). This leads to export of manganese and lowers intracellular manganese concentrations. Additionally, MntR downregulates the expression of MntH (blue) a manganese importer. When little manganese is present in the cell, this downregulation stops and manganese is transported into the cell. At the same time, the riboswitch prevents translation of mntP and manganese export by MntP is decreased.

A 10-min exposure to 10µM MnCl2 is sufficient for induction of the mntP transcript via this control mechanism [7]. Correspondingly, deletion of mntP or mntR results in heightened sensitivity to manganese and increased intracellular levels. Growth reduction of these phenotypes were specific to manganese, as inhibition could not be reproduced by other metals like zinc, magnesium, iron, nickel, or copper [7].

References

Terpe K. Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 2006; 72(2):211–22.
Qi X, Zhang Y, Chai T. Characterization of a novel plant promoter specifically induced by heavy metal and identification of the promoter regions conferring heavy metal responsiveness. Plant Physiol 2007; 143(1):50–9.
Arsène F, Tomoyasu T, Bukau B. The heat shock response of Escherichia coli. International Journal of Food Microbiology 2000; 55(1-3):3–9.
Babu KR, Swaminathan S, Marten S, Khanna N, Rinas U. Production of interferon-alpha in high cell density cultures of recombinant Escherichia coli and its single step purification from refolded inclusion body proteins. Appl Microbiol Biotechnol 2000; 53(6):655–60.
Singh AK, Sad K, Singh SK, Shivaji S. Regulation of gene expression at low temperature: role of cold-inducible promoters. Microbiology (Reading) 2014; 160(Pt 7):1291–6.
Chou CH, Aristidou AA, Meng SY, Bennett GN, San KY. Characterization of a pH-inducible promoter system for high-level expression of recombinant proteins in Escherichia coli. Biotechnol Bioeng 1995; 47(2):186–92.
Waters LS, Sandoval M, Storz G. The Escherichia coli MntR miniregulon includes genes encoding a small protein and an efflux pump required for manganese homeostasis. J Bacteriol 2011; 193(21):5887–97.
Patzer SI, Hantke K. Dual repression by Fe(2+)-Fur and Mn(2+)-MntR of the mntH gene, encoding an NRAMP-like Mn(2+) transporter in Escherichia coli. J Bacteriol 2001; 183(16):4806–13.
Dambach M, Sandoval M, Updegrove TB, Anantharaman V, Aravind L, Waters LS et al. The ubiquitous yybP-ykoY riboswitch is a manganese-responsive regulatory element. Mol Cell 2015; 57(6):1099–109.

@@ Line 9: / Line 9: @@
 ==Contribution by iGEM Team Tuebingen 2020==
-Author: Lea Vogt, Benedikt Jäger, Lina Widerspick
+Group: Team Tuebingen 2020
+Author: Lea Vogt, Lina Widerspick
+Summary:
 ===Usage===
@@ Line 21: / Line 25: @@
 <html><p><img src="https://2020.igem.org/wiki/images/9/96/T--Tuebingen--Manganese_Regulation.png" alt="Manganese Level Regulation" width="600px"></p></html>
-'''Figure 1: Regulation of manganese levels in ''E. coli.'' ''' During high cellular manganese levels the regulators MntR (yellow) and Fur activate the promoter (green) of the mntP gene (purple). This allows for transcription, but binding of manganese to the riboswitch (orange)  is necessary for following translation and expression of the manganese efflux pump MntP (purple). This leads to export of manganese and lowers intracellular manganese concentrations. Additionally, MntR downregulates the expression of MntH (blue) a manganese importer. When little manganese is present in the cell, this downregulation stops and manganese is transported into the cell. At the same time, the riboswitch prevents translation of mntP and manganese export by MntP is decrease
+'''Figure 1: Regulation of manganese levels in ''E. coli.'' ''' During high cellular manganese levels the regulators MntR (yellow) and Fur activate the promoter (green) of the mntP gene (purple). This allows for transcription, but binding of manganese to the riboswitch (orange)  is necessary for following translation and expression of the manganese efflux pump MntP (purple). This leads to export of manganese and lowers intracellular manganese concentrations. Additionally, MntR downregulates the expression of MntH (blue) a manganese importer. When little manganese is present in the cell, this downregulation stops and manganese is transported into the cell. At the same time, the riboswitch prevents translation of mntP and manganese export by MntP is decreased.
 A 10-min exposure to 10µM MnCl2 is sufficient for induction of the mntP transcript via this control mechanism [7]. Correspondingly,  deletion of  mntP or mntR results in heightened sensitivity to manganese and increased intracellular levels. Growth reduction of these phenotypes were specific to manganese, as inhibition could not be reproduced by other metals like zinc, magnesium, iron, nickel, or copper [7].