Difference between revisions of "Part:BBa K2123108"

Latest revision as of 08:06, 28 October 2016

Improved Mer Operon Device with novel regulated promoter

Overview

In 2014, our team constructed a synthetic genetic circuit which reached 74% of mercury bioremediation. Now, this composite part was developed to improve the mer operon expression, related to mercury metabolism, aiming to increase Hg. In order to do so, we designed 8 new promoters regulated by mer operon system. This composite part is the final device with the best of the regulated promoters. We achieved 97% of mercury bioremediation and validated under real world conditions, on the first bioreactor to Hg bioremediation. You can see the promoter characterization here: http://2016.igem.org/Team:UFAM-UEA_Brazil/Project/NewPromoters

Structure and mechanism:

This part is constituted by MerA, MerB, MerR, MerP and MerT.

MerA codifies the enzyme mercuric ion reductase, which catalyzes the reduction of mercuric ion Hg(II) to its volatile and non toxic form, Hg0. The active site of this enzyme has four cysteine residues that engage in mercury binding. The C-Terminal site is responsible for mercury catching from solution and delivering it to the core. This gene is the heart of mercury resistance in bacteria and its cytosolic protein works alongside a NADPH molecule, which offers the reductive power to reduce Hg(II) to Hg0.

MerB wides the range of mercuric forms that bacteria can be resistant to. MerB is translated into organomercurial lyase, an enzyme able to break the bonds between Hg and an organic radical, releasing Hg(II) to be reduced by MerA. This step is essential to fully sanate mercury contamination, since organomercury can be bound to living tissues and be biomagnified through food chain till it reaches humans.

MerR is the regulatory gene with its own promoter. MerR codifies the regulatory protein that binds the promoter region of the operon, in such a way that the recognition site of the promoter remains inaccessible to RNA polymerase, thus the operon's genes aren't translated. When mercury enters the cell, it binds to the C-terminal domain, causing an allosteric change on the protein's structure that propagates to N-terminal site, which is covering the promoter. This change causes the regulator to unwind DNA strand, allowing RNA polymerase to recognize the promoter and start the operon's functioning.

Mer P is a periplasmatic transporter protein, able to capture mercury organic or inorganic forms in periplasmatic space and deliver it to the membrane integrate protein, encoded by MerT gene. A pair of cysteine residues take mercury and its sulfer atoms ruptures the bonds between mercury and other binders, whose charge may be repeled by bacteria.

MerT protein, then receives mercury from MerP, at its first transmembrane helix. A pair of cysteine residues forms a complex with one cysteine from MerP, constituting mercury's deliver system. Then, another pair of cysteine, lying at cytoplasmatic face of MerT, receives Hg from the first helix and gives it directly to MerA protein, coupling mercury transport to the reduction mechanism.

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 below, 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). So, to validate it, our team constructed the first real bioreactor for mercury bioremediation of iGEM! See the results below!

After 18h, our construction reached 70% of mercury bioremediation! Want to see more? Access our wiki: 2016.igem.org/Team:UFAM-UEA_Brazil.

Sequence and Features