Team:LZU-China/chz project

Team:LZU 2015

 

This is our Project[2]

(subtitle)this is our principle

 

 

Background

Water pollution

Water, such a vital material for all kinds of creatures, plays a rather important role in human’s life. In China, Laozi, a well-known philosopher in the Spring and Autumn Period, thought highly of water and said,
‘The best of men is like water;
　　Water benefits all things.
　　And does not compete with them.
　　It dwells in (the lowly) places that all disdain -
Wherein it comes near to the Tao.’[1]
However, in our daily life, although water is noble, it is not so strong and usually gets sick. With the development of industry and modern society, water is badly polluted. So what is water pollution? Water pollution happens when toxic substances enter water bodies and degrade the quality of water. Although over 70% of the earth is covered by water, fresh water, which can be used by humans, occupied only 2.53% of the global water resources. Over 80% of rivers in China have some degree of contamination[2]

Some picture about water pollution reference from the Internet.

Water pollution can be classified into organic, inorganic and so on. What we care most is heavy metal pollution because heavy metal waste water is the current outstanding problem in the field of water pollution and becomes the focus of research in the controlling field.

Heavy metals from industrial processes can accumulate in nearby water body, which are toxic to marine life such as fish and shellfish, and subsequently to the humans who eat them. Heavy metals can slow development, result in birth defects and some are carcinogenic. The most commonly encountered toxic heavy metals in wastewater include Arsenic, Lead, Mercury, Cadmium, and the less common include Chromium, Copper, Nickel, Zinc[3]. Three kinds of heavy metals are of concern, including toxic metals (such as Hg, Cr, Pb, Zn, Cu, Ni, Cd, As, Co, Sn, etc.), precious metals (such as Pd, Pt, Ag, Au, Ru etc.) and radionuclides (such as U, Th, Ra, Am, etc.)[4]. Heavy metals bring about serious environmental pollution, threatening human health and ecosystem.[5]

here are millions people are struggling with water pollution. In China, for example, about 75 percent of the population (or 1.1 billion people) are without access to unpolluted drinking water, according to standards from China[6]. And our university, Lanzhou University, located on northwestern China, where lacking water especially. What’s worse, water pollution is also serious including heavy metal contaminants.

MFC

Instead of exploiting more water resources, which has more and more negative influence[1], it’s better to make less pollution and treat water pollution.[2] And we must know what kinds of contaminations in the water and how much are them. So we chose MFC (Microbial Fuel Cells) to detect the heavy metal in water.

So, what is MFC?

Microbial fuel cells (MFCs) provide new opportunities for the sustainable production of energy from biodegradable, reduced compounds. MFCs function on different carbohydrates but also on complex substrates present in wastewaters，which is an ideal approach to solve both pollution problem and energy crisis. Compared with traditional chemical methods of sewage treatment, MFC is environmental friendly and widely adept by various condition[3].

A MFC converts biomass energy directly into electricity. This can be achieved when bacteria switch from the natural electron acceptor, such as oxygen or nitrate, to an insoluble acceptor, such as the MFC anode. A typical microbial fuel cell consists of two compartments: anode and cathode compartments separated by a proton exchange membrane (PEM). In the anode compartment, microorganisms oxidize the fuel and generating CO2, electrons and protons. Electrons are transferred through an external electric circuit to the cathode compartment, while protons are transferred to the cathode compartment through the membrane. Electrons and protons are consumed in the cathode compartment, combining with oxygen to form water.

Figure 1. The working principle of a microbial fuel cell. Substrate is metabolized by bacteria, which transfer the gained electrons to the anode. This can occur either directly through the membrane or via mobile redox shuttles. MED, redox mediator; Red oval, terminal electron shuttle in or on the bacterium.[4]

After electron generated, the electron need to be transferred out of the microbe, The mechanism includes two methods:
1.Direct transfer mechanism: Electrons are directly transferred from the microbe’s cell membrane to the anode surface（A）、nanowires transfer the electron through conductive appendages, termed “nanowires”, grown by the bacteria.（B）
2.Indirect transfer mechanism: Microbes employ a secondary biomolecule——electron transfer mediators（ETMs） to shuttle the electron to the anode（C）.

Figure2: Schematic diagram of extracellular electron transfer mechanisms for electrochemically active microorganisms in anode@

Although MFC is high efficiency of power output and environment friendly, most of the power generation MFCs are still in laboratory and not come into massive scale of industry.

One major drawback is the output power density is far less to adapt the standard in industry. electron transfer rate is determined by the potential difference, the reorganization energy and electron donor and receptor distance, main factors to determine the output power density of the microbial fuel cell is related to the electron transfer process, that is to say, the biological system of slow electron transfer rate is the bottleneck of the development of microbial fuel cell.

  • 1. Hou-gui, Z., Environmental issues and countermeasures in exploiting water resources of rivers. Journal of Chongqing University 2006. 5(2): p. 111-114.
  • 2. Rabaey, K. and W. Verstraete, Microbial fuel cells: novel biotechnology for energy generation. Trends in Biotechnology, 2005. 23(6): p. 291-298.
  • 3. Liu, L., et al., Electron Transfer Mediators in Microbial Electrochemical Systems. PROGRESS IN CHEMISTRY, 2014(11): p. 1859-1866.
  • 4. Chouler, J. and M. Di Lorenzo, Water Quality Monitoring in Developing Countries; Can Microbial Fuel Cells be the Answer? Biosensors, 2015. 5(3): p. 450.
Riboflavin

Riboflavin, a yellow-orange solid substance with poor solubility in water, was originally recognized as a growth factor in 1879 and named vitamin B, according to the British nomenclature system.

Its IUPAC name is 7,8-Dimethyl-10-[(2S,3S,4R)-2,3,4,5-tetrahydroxypentyl]benzo[g]pteridine-2,4-dione[8]. Riboflavin has two active coenzyme forms, riboflavin 5'-phosphate (R5P; flavin mononucleotide [FMN]) and flavin adenine dinucleotide (FAD)[9], which can both transfer electron.

Fig 1 Riboflavin: C17H20N4O6. Reference: https://en.wikipedia.org/wiki/Riboflavin

As riboflavin’s structure shows, there is a transfer of two electrons from hydrogen and hydrid ions, so riboflavin can be regarded as a electron shuttle and even a kind of redox mediator. Though riboflavin is very important in mediator-driven bioelectricity generation, the detailed mechanism on electron transfer has not been precisely investigated yet[10].

Researchers find that increase of riboflavin biosynthesis can profounfly enhance extracellular electron transfer in bacteria and then improve the efficience of MFC. For example, the increase of riboflavin biosynthesis by Shewanella at the alkaline condition underlies the improvement of the electricity output in MFCs(Fig 2)[11].

Fig 2 Increased riboflavin synthesis underlies enhanced electron transfer in Shewanella. We draw the conclusion that increased riboflavin synthesis can improve the MFC’s production.

So riboflavin became our star. Since it can be expressed in bacteria, we can combine riboflavin and MFC to improve the electricity output of MFC. And it is easy and mature to measure the concentration of rirboflavin in aqueous solution based on the technique of OD (optical density). The peaks of absorbance occur at 223, 266, 373 and 445 nm(Fig 3)[12]. P. Drossler etc. investigated a wide range of pH values from pH -1.1 to pH 13.4 and made the absorption spectrum curve over different pH value(Fig 4)[13].

Fig 3 Absorption spectrum of riboflavin in aqweous solution. And the peaks of absorbance occur around at 223, 266, 373 and 445 nm
Fig 4 Absorption cross-section spectra of riboflavin in aqueous solutions at various values of pH. The riboflavin concentration used in the measurements was around 10-4 mol dm3

In our laboratory, we used the multi-spectral scanner to analyse the optimal absorption wavelength and finally found absorption peak at 444nm is the optimum in pH 1.17. And our optimal absorption wavelength corresponds with previous researches. In order to make pH 1.17, we mixed 2ml 0.1mol/L HCl with 1ml riboflavin aqueous solutions extract from engineering bacteria. Then it is able to detect the concentration of rirboflavin by OD value.

There are many methods to get riboflavin by microorganism. In E.coli, a gene cluster, consisting of RibA, RibB, RibDG, RibH and RibE and separating to different transcription units, regulates the synthesis of riboflavin. The details of riboflavin biosynthesis have been figured out(Fig 5)[14].

Fig 5 Biosynthesis of riboflavin and flavocoenzymes from Fischer, 2006 [10]. Step A, GTP cyclohydrolase III; step B, GTP cyclohydrolase II; step C, 2-amino-5- formylamino-6-ribosylamino-4(3H)-pyrimidinone 5’-phosphate hydrolase; step D, 2,5- diamino-6-ribosylamino-4(3H)-pyrimidinone 5’-phosphate deaminase; step E, 5- amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5’-phosphate reductase; step F, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5’-phosphate reductase; step G, 2,5- diamino-6-ribitylamino-4(3H)-pyrimidinedione 5’-phosphate deaminase; step H, hypothetical phosphatase; step I, 3,4-dihydroxy-2-butanone 4-phosphate synthase; step J, 6,7-dimethyl-8-ribityllumazine synthase; step K, riboflavin synthase; step L, flavokinase; step M, FAD synthetase; 1, GTP; 2, 2,5-diamino-6-ribosylamino-4(3H)- pyrimidinone 5’-phosphate; 3, 2-amino-5-formylamino-6-ribosylamino-4(3H)- pyrimidinone 5´-phosphate; 4, 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5’-phosphate; 5, 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5’-phosphate; 6, 5- amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5’-phosphate, 7; 5-amino-6- ribitylamino-2,4(1H,3H)-pyrimidinedione; 8, ribulose 5-phosphate; 9, 3,4-dihydroxy-2- butanone 4-phosphate; 10, 6,7-dimethyl-8-ribityllumazine; 11, riboflavin; 12, FMN; 13, FAD.

Green arrows mark the plant pathway; red, fate of the four-carbon precursor 9 derived from ribulose 5-phosphate.

  • 1. Laozi, Tao Te Ching. 516 BC. 8.
  • 2. Qi, F., C. Guodong, and M. Masao, Water Resources in China: Problems and Countermeasures. Ambio, 1999. 28(2): p. 202-203.
  • 3. Heavy metals in wastewater. 2013.
  • 4. Wang, J. and C. Chen, Biosorption of heavy metals by Saccharomyces cerevisiae: A review. Biotechnology Advances, 2006. 24(5): p. 427-451.
  • 5. Wang, J. and C. Chen, Biosorbents for heavy metals removal and their future. Biotechnology Advances, 2009. 27(2): p. 195-226.
  • 6. Hanson Ii, J.R., The World Economy: A Millennial Perspective (Book). Journal of Economic Literature, 2002. 40(4): p. 1256-1257.
  • 7. Hou-gui, Z., Environmental issues and countermeasures in exploiting water resources of rivers. Journal of Chongqing University, 2006. 5(2): p. 111-114.
  • 8. PubChem 493570.
  • 9. Riboflavin. Alternative Medicine Review, 2008. 13(4): p. 334-340.
  • 10. Jung, S.-H., et al., Impedance and Thermodynamic Analysis of Bioanode, Abiotic Anode, and Riboflavin-Amended Anode in Microbial Fuel Cells. Bulletin of the Korean Chemical Society, 2012. 33(10): p. 3349-3354.
  • 11. Yong, Y.C., et al., Increase of riboflavin biosynthesis underlies enhancement of extracellular electron transfer of Shewanella in alkaline microbial fuel cells. Bioresour Technol, 2013. 130: p. 763-8.
  • 12. Lu, C., et al., Photophysical and photochemical processes of riboflavin (vitamin B2) by means of the transient absorption spectra in aqueous solution. Science in China Series B: Chemistry, 2001. 44(1): p. 39-48.
  • 13. Drössler, P., et al., pH dependence of the absorption and emission behaviour of riboflavin in aqueous solution. Chemical Physics, 2002. 282(3): p. 429-439.
  • 14. Kim, R., Biosynthesis of Vitamin B2 (Riboflavin) Studies on the Reaction Mechanism of Riboflavin Synthase. 2012.

 

Wet Lab

Introduction

As it is known to all, Sherlock Holmes is a famous detective. Based on synthesis biology, our team creates a smart system called Micro Holmes which can monitor the pollutant inside the water in real time. Once the system was founded, through users’ laptops, smartphones or some other media, we would acquire the statistics of pollutant which has been analyzed by the system. Reforming the E.Coli, we endowed them with the ability of sensing the pollutant inside the water and delivering the message of it. Therefore, raising them inside the Microbial Fuel Cell (MFC), they can rapidly augment the voltage as soon as the polluted occurred. Then, by the automatically collecting and analyzing equipment, we can know the concentration of pollutant converted by the instant electric signal. Escherichia coli, a tiny but full of power creature, perfectly plays an important part as Holmes in our project. So, are you interest in how he was created and how he works?

First of all，Mr. Holmes must has a piercing eye to find the trail of pollutant; And after detecting pollutant, Mr. Holmes must has the ability to convey the message to us in time. Therefore, in order to constitute a series of inductivity parts—which can be efficiently expressed in E.Coli BL 21 bacterial strains—we link the efficient receptor of heavy metal ions with RibB--- the key synthesis gene of riboflavin. They can translate the information from the concentration of heavy metal ions to the riboflavin content, which is the product of reporter gene RibB. Riboflavin is a kind of redox mediator who works as a highly-efficient electronic transfer mediator. When the concentration of Riboflavin increased, it can improve the efficiency of electronic translocation between E.Coli and the anode and eventually augment the voltage. Combined with MFC (microbial fuel cell) system, we can measure the concentration of heavy metal ions in water through the voltage presented by MFC system and realize the conversion of heavy metal ions concentration to electrical signal. This method has lots of advantages, such as compatibility, high efficiency, instantaneity, reliability and so on.

The main idea of designing this experiment can be seen in the following flow chart.

Fig 1 Whole Experiment Flow Chart

In this project, parts were divided into two kinds. One is constitutive part, combined by various type of promoter, RBS, RibB and terminator. We constructed the constitutive parts to select a highly-expressed part of RibB. The other is inducible part with RBS, RibB terminator and promoter which can sense heavy metal ions. This kind of parts can highly express RibB so that it is practical to associate the concentration of heavy metal ions with the output of riboflavin. Therefore, putting the E.Coli BL 21 into the MFC, we collected the statistics of MFC on the condition that concentration of metal ion solution was already known. So we built the correlation model, then induced the E.Coli BL21 with the solution to be measured. It is easy to figure out the concentration by measuring the voltage of MFC. We can monitor the specific metal ions real-timely, quantitatively and swiftly through this way and more importantly, it has compatibility in the measurement of metal ion by changing the parts to detect different metal ions.

 

An efficient riboflavin synthesis gene: RibB
1 An Introduction of RibB
1.1 The biosynthesis of riboflavin inside E.coli

Riboflavin, also called vitamin B2, can be synthesized by various kinds of plants and animals. The basic synthesis principal is similar, that one molecular of GTP and two molecular of ribulose 5-phosphate undergo a series of enzymatic reaction[1]. However, the operons vary from bacteria to bacteria. At present, we have made the principal clear in most kind of bacterium and in this project, we chose E.coli in which the riboflavin synthetic genes emerge as nonlinear gene cluster and was distributed into nonhomologous chromosome.

Fig 2-A shows the process of riboflavin synthesis and its relevant genes including RibA (coding GTP cyclohydrolase II), RibB (coding 3,4-dihydroxy-2-butanone-4-phosphate synthase), RibDG (coding a kind of bifunctional riboflavin specificdeaminase/reductase), RibH (coding lumazine synthase) and RibE (coding riboflavin synthase). The distribution of these gene can be seen in Fig 2-B[2].

Fig 2-A The riboflavin biosynthesis inside Escherichia coli[2].

Fig 2-B RibA, RibB, RibDG, RibH and RibE inside the E.coli are not on the same transcription unit but separate to different transcription units which have different promoters. Genes in yellow are related to the synthesis of riboflavin while the grey are not known to be involved in flavin biosynthesis[2].

1.2 RibB and its function

The synthesis of riboflavin is complicated because there are so many genes involved in this process. Besides, those genes are not focus on a same operon. According to some research, RibB is of vital importance to the synthesis of riboflavin which in charge of coding DHBP synthetase and can catalyze the synthesis of 3,4-dihydroxy-2-butanone-4-phosphate[3]. Researches conducted by Danielle Pedrolli and other researchers show that the FMN riboswitch, which is in the untranslated region of 5’ terminal of RibB, can regulate the expression of RibB so that it can inhibit the biosynthesis of riboflavin. Thus, the enzyme translated by RibB becomes a limiter of riboflavin synthesis. Therefore, we can evidently improve the output of riboflavin by knocking out the FMN riboswitch or overexpressing RibB. Related experiment statistics can be referred from Fig 3. Compared with the whole riboflavin synthesis genes cluster, RibB has the advantages of convenience, high efficiency and short sequence as it is only 654bp. Consequently, our team choosed RibB as a report gene to construct parts and then builded the connection between heavy metal ions and RibB and finally used the product of RibB—riboflavin to impact the voltage of MFC so that we realized the monitoring of heavy metal ions automatically, quantitatively and efficiently.

Fig 3 The expression of riboflavin increased[2]. Fig 3-A After knocking out the FMN riboswitch, the expression of riboflavin increased. Abbreviation “del”, the test tube on the right side, is the E.coli knocked out the FMN riboswitch and the pNCO113 is control.
Fig 3-A After knocking out the FMN riboswitch, the expression of riboflavin increased. Abbreviation “del”, the test tube on the right side, is the E.coli knocked out the FMN riboswitch and the pNCO113 is control.
Fig 3-B After overexpressing RibB, the expression of riboflavin increased. “ribB”, the test tube on the right side, is the E.coli overexpressed RibB and the pNCO113 is control.

2 The measurement data of several constitutive parts

Totally, we constructed 5 kinds of constitutive parts and quantitatively measured their ability of producing of riboflavin. The character of these 5 parts can be seen in Table 1. Table 1 Part of the message of measured part

NO. Construction
K1755005 K608003(strong promoter +medium RBS)+ ribB + B0014(terminator)
K1755006 K608006(medium promoter + medium RBS) + ribB + B0014(terminator)
K1755007 K608002(strong promoter +strong RBS) + ribB + B0014(terminator)
K1755008 K608007(medium promoter +weak RBS) + ribB + B0014(terminator)
K1755009 K608004(strong promoter +weak RBS) + ribB + B0014(terminator)

We cultured the transgened E.coli BL21 in M9 colorless culture media while at the same time set up a control with the original BL21. Compared with the control, it is seemingly that the BL21 with RibB gene part can product more riboflavin (riboflavin solution looks yellow), seeing Fig 4.

Fig 4 From left to right, respectively, the solution is BL21 control, BL21 with K1755005, BL21 with K1755006, BL21 with K1755009.
Use spectrophotometer to measure the concentration of bacteria and riboflavin at different time. The consequence of the experiment can be seen in Fig 5.

Figure5-A
 

Fig 5-A Bacteria concentration of K1755005, K1755006, K1755009 bacterial strains vary with time.

Concentration of the microbial strains of K1755005, K1755006, K1755009 bacterial strains basically don't have difference at the same point in time, and the corresponding statistical analysis also showed that there are no significant differences between the three parts(the results are shown in Table 2). It shows that when bacterial strains are linked with K1755005, K1755006 or K1755009, the influence to the viability of the bacteria is very limited and can even ignore it. That's why the concentration of the microbial strains is basically the same with the different processing at the same time. In the following research we can directly compare the concentration of riboflavin to analyze the ability of riboflavin synthesis in different bacterial strains.

Figure5-B
 

Fig 5-B Riboflavin concentration of K1755005, K1755006, K1755009 bacterial strains vary with time.

The bacterial strain linked with RibB has a better performance of producing riboflavin and the promoting ability from strong to weak can be easily seen as following: K1755005, K1755009 and K1755006. Moreover, the distinction between K1755009 and K1755006 isn't obvious. The construction of these three parts are familiar and the the statistical analysis also showed that when the bacterial strain linked different parts, the ability of producing riboflavin is totally different with the original BL21 (The analysis result is shown in table 2), which proved that K1755005, K1755009 and K1755006 have all accomplished as we anticipated.

Figure5-C
 

Fig 5-C Bacteria concentration of K1755007 and K1755008 bacterial strains vary with time.

Although there are some difference in bacteria density between strain K1755007 and strain K1755008 at the same time, the difference isn't obvious. Statistical analysis also shows that the difference of bacteria density between K1755007 and control group isn't obvious, but the difference of bacteria density between K1755008 and control group is remarkable.

Figure5-D
 

Fig 5-D Riboflavin concentration of K1755007 and K1755008 bacterial strains vary with time.

After linking different parts, riboflavin expression level is distinctly different because K1755007 is constructed with strong promoter and strong RBS so that it has the strongest ability to produce riboflavin followed by K1755008, which is weaker than K1755007. The experimental phenomena coincide with theoretical analysis, showing that K1755007 and K1755008 both can work. Some problematic data have been removed.

Figure5-E
 

Fig 5-E The ratio of riboflavin and bacteria concentration of different kinds of BL21 vary with time.

Through the ratio curve of riboflavin and OD value from the same serial number and the same time point, we can intuitively know that K1755007 and K1755008 can observably improve riboflavin production in E.Coli and K1755007 behaves batter than K1755008.

Using SPSS (Statistical Product and Service Solutions) paired sample t test for significance test, we obtain the following results on the condition that the confidence level is 95%. Table 2 Significance test between different part and BL21

  • A
    • A
      dada
    • B
    • C
NO. Bacteria concentration Riboflavin concentration
Alvin Eclair $0.87
Alan Jellybean $3.76
Jonathan Lollipop $7.00
2.3 The relationship between riboflavin and produce electricity

Studies have shown that riboflavin can observably increase the voltage of MFC. By doing the experiment, we also confirmed that riboflavin can indeed improve the production capacity. And according to relevant data, we drew graph 5 (the data in this graph is measured in last year, the latest data is still in the measurement).

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Fig 6 The effects of riboflavin on voltage.

Through the significant changes in the voltage of MFC before and after adding riboflavin, we can know that riboflavin can improve the voltage of MFC obviously.

  • 1. Fischer, M. and A. Bacher, Biosynthesis of vitamin B2: Structure and mechanism of riboflavin synthase. Arch Biochem Biophys, 2008. 474(2): p. 252-65.
  • 2. Pedrolli, D., et al., The ribB FMN riboswitch from Escherichia coli operates at the transcriptional and translational level and regulates riboflavin biosynthesis. FEBS J, 2015. 282(16): p. 3230-42.
  • 3. Lin, J.W., Y.F. Chao, and S.F. Weng, Riboflavin synthesis genes ribE, ribB, ribH, ribA reside in the lux operon of Photobacterium leiognathi. Biochem Biophys Res Commun, 2001. 284(3): p. 587-95.
Pollutant sensor

1 Cu2+ sensor(& Cr6+sensor)

1.1 BBa_K1755301

1.1.1 The composition of BBa_K1755301

We connected BBa_K1755401（MarO）, as a promoter, with BBa_1755003（RBS+ribB CDS+B0014）to get BBa_K1755301.

1.1.2 How does it work

MarO is a promoter, which belongs to the mar operon in E.coli. Genetic organization of the mar regulon in E.coli shows as Fig 2a. There are two promoter sites on the marO operator, and both of them can be bound by a kind of regulatory protein called MarR, which is coded by MarR and can repress the expression of marO(Fig 2b and Fig 2c)[2]. MarR is a very special and vital transcription factor, which can bound to either of marO’s two promoter sites rs1 and rs2(rs means repressor binding site). And MarR repressor is homodimer. And it can form tetramer once there is copper for its special structure(Fig 3). Copper(II) oxidizes a cysteine residue (Cys80) on MarR to generate disulfide bonds between two MarR dimers, thereby inducing tetramer formation and the dissociation of MarR from its cognate promoter DNA, two sites of marO(Fig 4)[1]. Some experiments firmed the explanation(Fig 5)[1].

So we chose promoter marO as the copper sensor. Cu2+ can oxidize Cys80 and make MarR apart from marO to derepress the MarR dimer. Than the promoter marO will start the following genes’ transcription. In our part BBa_K1755301, the report gene is riboflavin. So we associated the copper concentration with the output of riboflavin.

During our experiments, it is a big surprise for us to find that the marO is able to respond to Cr6+ as well though it is not so strong as Cu2+.

Fig 2a Genetic organization of the mar operon in E.coli.
Fig 2b The products of marR can repress the function of marO in E.coli
Fig 2c The 2 sites of promoter marO in E.coli
Fig 3 Crystal structure of copper(II)-oxidized MarR5CS(80C).
(a) Copper(II)-oxidized MarR5CS(80C) tetramer with two monomers from each dimer (chain A–chain B and chain A’–chain B’) colored blue and magenta, respectively. Atoms in the side chains of Cys80 from all four of the monomers are shown as yellow spheres. (b) Close-up of the two disulfide bonds between two MarR dimers. The disulfide bonds shown in yellow sticks. Only residues Asp67(Asp67’), Cys80(Cys80’) and Gly82(Gly82’) from each monomer are shown for clarity. (c) Superposition of the structures of copper-oxidized MarR5CS(80C) (blue) and the previously reported SAL-complexed WT MarR(orange).
Fig 4 The mechanism of Cu2+ derepress of MarO
Fig 5 Some experiments results verified the explanation of the mechanism of MarO[1]
1.1.3 Results
We linked BBa_K1755301 into pSB1C3 and transformed the new plasmid into E.coli BL-21. Then we detected the concentration of riboflavin through measuring the absorbance under 444nm and found that riboflavin production basically response to the change of metal ion concentration as it present a linear relationship with Cu2+ concentration, which indicated that BBa_K1755301 can work as we anticipated.

1.2 BBa_K1755302

1.2.1 The composition of BBa_K1755302

We connected BBa_K190024（CueO+RBS）, as a, with BBa_1755002（ribB CDS+B0014）to get BBa_K1755302. The former part is a sensor and the latter is a reporter.

1.2.2 How does it work

CueO can express periplasmic copper-binding proteins, CueO , a multi-copper oxidase for detoxification[1], which belongs to CueR Regulon gene and can be regulated by copper-responsive transcription factor CueR, too. CueO can oxidate Cu+ to Cu2+ and decrease the toxicity of copper[3, 4]. On the CueO promoter, CueR protectes -41 to -17 on top strand and -42 to -19 on bottom strand to repress the expression of CueO[1]. And it can be derepressed by Cu2+, too. So the CueO promoter can sense the concentration of copper and linkage it with the production of riboflavin.

1.2.3 Results

We linked BBa_K1755302 into pSB1C3 and transformed the new plasmid into E.coli BL-21. Then we detected the concentration of riboflavin through measuring the absorbance under 444nm and found that riboflavin production basically response to the change of metal ion concentration as it present a linear relationship with Cu2+ concentration, which indicated that BBa_K1755302 can work as we anticipated.

2 Hg2+ sensor

2.1 BBa_K1755305

2.1.1 The composition of BBa_K1755305

We connected BBa_K346002（PmerT）, as a promoter, with BBa_1755003（RBS+ribB CDS+B0014）to get BBa_K1755305.

2.1.2 How does it work

PmerT is a kind of promoter from Tn21 mercury resistance (mer) operon. When there is no Hg2+, it cannot start the transcription. Upon binding the cognate metal ions, the metallated MerR homodimer causes a realignment of the promoter so that RNA polymerase contacts the -35 and -10 sequences leading to open complex formation and transcription. (see more details http:// parts.igem.org /Part:BBa_K346002).

So PmerT promoter can be induced by Hg2+ and start the coding of ribB downstream to increase the output of riboflavin. And there is some quantity relationship about the concentration of Hg2+ and the production of riboflavin. We can calculate the the concentration of Hg2+ by our “Micro Holmes” system.

2.1.3 Results

We transformed BBa_K1755305 into E.coli BL-21. Then we detected the concentration of riboflavin through measuring the absorbance under 444nm. Unfortunately, we didn’t find that riboflavin production respond to the change of Hg2+ concentration, which indicated that BBa_K1755305 can not work as we anticipated.

3 F- sensor

3.1 BBa_K1755024

3.1.1 The composition

We used BBa_K911003 as a promoter and connected it with BBa_1755003（RBS+ribB CDS+B0014）to get 3.1 BBa_K1755024.

3.1.2 How does it work

BBa_K911003 is a fluoride sensitive riboswitch that is highly sensitive to the F-. It is a positive regulator so F- can induce the promoter to transcript. Since the report gene is at the downstream of fluoride promoter, the riboflavin production can be influenced by the change of F- concentration. So we can use the concentration of riboflavin to determine the concentration of F- and even apply into our “Micro Holmes” system.

3.1.3 Results

We transformed BBa_K1755024 into E.coli BL-21. Then we detected the concentration of riboflavin through measuring the absorbance under 444nm. However, we didn’t find regular change relationship between riboflavin production and Hg2+ concentration. The result showed that BBa_K1755024 can not work as we anticipated.

  • 1. Hao, Z., et al., The multiple antibiotic resistance regulator MarR is a copper sensor in Escherichia coli. Nat Chem Biol, 2014. 10(1): p. 21-8.
    • 2. R., C.X.H.Z.C.P., Protein photocrosslinking reveals dimer of dimers formation on MarR protein in Escherichia coli. 中国科学：化学, 2012. 42(2): p. 223-225. 3. Yamamoto, K. and A. Ishihama, Transcriptional response of Escherichia coli to external copper. Mol Microbiol, 2005. 56(1): p. 215-27. 4. Stoyanov, J.V., J.L. Hobman, and N.L. Brown, CueR (YbbI) of Escherichia coli is a MerR family regulator controlling expression of the copper exporter CopA. Molecular Microbiology, 2001. 39(2): p. 502-512.

We connected BBa_K346002（PmerT）, as a promoter, with BBa_1755003（RBS+ribB CDS+B0014）to get BBa_K1755305.

2.1.2 How does it work

During our experiments, it is a big surprise for us to find that the marO is able to respond to Cr6+ as well though it is not so strong as Cu2+.

Our MFC

Dry Lab

Mrcro Holmes——Concentration Measurement System
An efficient riboflavin synthesis gene: RibB
An efficient riboflavin synthesis gene: RibB
An efficient riboflavin synthesis gene: RibB
An efficient riboflavin synthesis gene: RibB
An efficient riboflavin synthesis gene: RibB
An efficient riboflavin synthesis gene: RibB
An efficient riboflavin synthesis gene: RibB

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Figure1
Figure1

 

: China, as the world largest developing country, suffers serious pollution in 21 century. Lanzhou, a capital city in northwest of China, bears serious contaminations.

China, as the world largest developing country, suffers serious pollution in 21 century. Lanzhou, a capital city in northwest of China, bears serious contaminations.

China, as the world largest developing country, suffers serious pollution in 21 century. Lanzhou, a capital city in northwest of China, bears serious contaminations.