Difference between revisions of "Part:BBa K2123200"

Latest revision as of 09:37, 22 October 2016

Strong RBS + MerR (regulatory protein) + trp Terminator

Overview:

MerR is an integrate part of Mer Operon a set of genes that grant bacterial resistance to mercury. MerR gene codifies a regulatory protein which ties itself to mer operator (MerO) of the bidirectional promoter region, activating the mer operon expression and therefore the mercury resistance mechanism. In the absence of Hg, MerR is a weak repressor.

Structure and mechanism:

Molecular structure of MerR protein is constituted of 144 individuals amino acids that differ in nine type of residues. Structurally, the regulator presents itself as an homodimer with mass equivalent to 31 kDa. The C and N-terminal extremities are intimately connected in MerR role as regulator. C-terminal is the Hg binding site, mediated by three cysteine residues. Then, N-terminal is linked to the operator (MerO), between -35 and -10 regions, so the recognition site remains inaccessible to RNA polymerase and genes transcription does not begin. MerR is able to attract RNA polymerase to the promoter region, even in absence of Hg(II). The pre-transcriptional complex merR-polymerase remains steady and not producing until Hg(II) is bound to merR’s C site, causing an allosteric change in the protein’s structure. This is propagated to the DNA strain, which unwinds and favors RNA polymerase binding to the promoter, and transcription starts. The formation of pre-transcriptional complex allows quick answer to Hg(II) presence in the medium. The regulatory mechanism of merR and activation of Mer Operon is illustrated below:

Figure 01: Transcription activation of Mer Operon. A) Pre-transcriptional complex, composed by merR, RNA polymerase and the others transcription factors: merR conformity does not allows RNA polymerase recognition, thus Mer Operon remains off. B) Hg binding to terminal C of the regulator promotes conformational changes in the protein and allows RNA polymerase to starts transcription. As soon as Hg(II) is reduced to its volatile form (Hg0), Mer Operon needs to be repressed again. Hg-sulfur bonds settled with repressor is very steady and its unlikely that Hg leaves merR spontaneously. There are two hypothesis on how Mer Operon is repressed when mercury leaves the cell: a merR protein not complexed with Hg(II) bounds to regulation site and discontinues transcription; or the regulatory protein antagonist to merR, merD, takes the role of repressor and inactivates Mer Operon.

Sources:

BARKAY, T.; MILLER, S. M.; SUMMERS, A. O. Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiology Reviews 27 (2003) 355-384.

BROWN, N. L.; STOYANOV J. V.; KIDD, S. P.; HOBMAN, J. L. I. The MerR family of transcriptional regulators. FEMS Microbiology Reviews 27 (2003) 145-163.

O’HALLORAN, T. V.; FRANTZ, B.; SHIN, M. K.; RALSTON, D. M.; WRIGHT, J. G. The MerR heavy metal receptor mediates positive activation in a topologically novel transcription complex. Cell 56 (1989) 119-129.

Usage, Methodology and Experiments

Our team (UFAM-UEA_Brazil) worked improving the Mer operon expression to increase bioremediation in E. coli through novel mer promoters sequences. For it, we primarily characterized the MerR expression under control of different promoters from Anderson Collection (BBa_J23100, BBa_J23104, BBa_J23106 e BBa_I142033) through the repression of RFP (BBa_K081014) production, in a synthetic genetic circuit represented bellow.

On the first experiment, we used the novel regulated promoter designed by our team BBa_K2123109 (Stationary growth phase promoter with downstream mer operator) to measure the RFP repression by MerR under control of BBa_J23100, BBa_J23104, BBa_J23106 e BBa_I142033 constitutive promoters in solid LB media, as you can see below.

You can analyse by the RFP expression and thus fluorescence intensity that the samples greater repressed by MerR was the ones under control of BBa_J23100 and BBa_I14033 constituve promoters. So, we measured the RFP expression using Chamaleon Spectrofluorometer with and without MerR repressor protein under control of these two constituve promoter. The results are presented in the graph 1 below.

Graph 1: RFP expression with and without MerR regulator under control of BBa_J23100 and BBa_I14033. Control bacteria is DH5-alpha without any plasmid vector.

As we can see, between BBa_J23100 and BBa_I14033, the best repressed was with BBa_J23100 constitutive promoter. So, we selected BBa_J23100 to controls MerR expression in our synthetic genetic circuits. We made also other experiments to understand the interactions between MerR and new regulated promoters designed by our team, aiming to reach a well repressor mechanism, increasing the natural MerR regulation. In this way, we measured MerR regulation with two more regulated promoters: BBa_K2123102 and BBa_K2123101 - Tac promoter + overlapped and between mer operator, respectively expression RFP. The MerR regulation mechanism was characterized by repressing RFP expression and therefore reducing fluorescence intensity according to the regulator efficiency. We measured it also utilizing Chamaleon Spectrofluorometer. The results are presented in the graph 2 and 3 below.

Graph 02 and 03: RFP expression with and without MerR regulator under control of BBa_J23100 constitutive promoter, repressing BBa_K2123101 and BBa_K2123102 new regulated promoters. We achieved almost a totally repression with this BioBrick part! Awesome results, as you can see in the figures below.

Figure 04: RFP expression regulation by MerR.

To finalize this part characterization, we cloned it with the Mer Operon improved genes device (as the figure aiming to measure the amoung of mercury bioremediated under control of our MerR with very strong regulation in 7.5ppm of mercury chloride with 10 hours of growth.

Previous iGEM projects achieved 75% of mercury bioremediated with their synthetic genetic circuits. With our improvement, designing new regulated promoters sequences and thus increasing MerR regulation, this amoung increased to =97%= of mercury bioremediation in E. coli DH5-alpha. We measured with DMA-80 (Direct Mercury Analyser) equipment. Check this out below!

Graph 04: Comparison growth curve of previous synthetic genetic circuits (BBa_K1355004) with our improved devices in 7.5ppm of mercury chloride, measured with spectrophotometer (600nm wavelenght).

Graph 05: Amoung of mercury after 10 hours of bacterial growth with our construction (BBa_K2123108). Sequence and Features