Fluorescence expression profiling of whole-cell biosensors - a proposal for a new dual labeled biosensing system

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(1)FLUORESCENCE EXPRESSION PROFILING OF WHOLE-CELL BIOSENSORS. A PROPOSAL FOR A NEW DUAL LABELED BIOSENSING SYSTEM. A DISSERTATION SUBMITTED TO THE COMMITTEE ON GRADUATE SCHOOL OF BIOLOGICAL SCIENCES DEPARTMENT IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCES IN BIOLOGICAL SCIENCES. NICOLÁS CAICEDO HERNANDEZ ADVISOR: JENNY DUSSÁN GARZÓN Ph.D.. BOGOTÁ, JUNE 2009.

(2) TABLE OF CONTENTS ABSTRACT ...................................................................................................................... - 1 INTRODUCTION ............................................................................................................. - 2 1. MATERIALS AND METHODS ............................................................................ - 11 1.1. Reagents .......................................................................................................... - 11 1.2. Bacterial Strains, Plasmids and Growth Conditions........................................ - 11 1.3. Gene-Splicing by Overlap Extension PCR...................................................... - 13 1.3.1. First PCR step .......................................................................................... - 14 1.3.2. Second PCR step ..................................................................................... - 15 1.3.3. Third PCR Step........................................................................................ - 16 1.4. Construction of the Biosensor Strains ............................................................. - 16 1.4.1. Single-labeled .......................................................................................... - 16 1.4.2. Dual-labeled ............................................................................................ - 16 1.5. Compound Response Assays ........................................................................... - 17 1.5.1. Lead ......................................................................................................... - 17 1.5.2. Salicylate ................................................................................................. - 17 1.5.3. Selectivity Tests....................................................................................... - 18 1.6. Fluorescence Assays ........................................................................................ - 18 1.7. RNA Isolation .................................................................................................. - 18 1.7.1. DNase Treatment ..................................................................................... - 18 1.8. Reverse-Transcription quantitative PCR ......................................................... - 19 1.8.1. Calibration Curves and qPCR Standards ................................................. - 20 1.9. Protein Electrophoresis and Western Blotting ................................................ - 21 1.10. Fluorescence Microscopy ............................................................................ - 21 2. RESULTS AND DISCUSSION.............................................................................. - 22 2.1. Gene-Splicing by Overlap Extension PCR...................................................... - 22 2.1.1. First PCR step .......................................................................................... - 22 2.1.2. Second PCR step ..................................................................................... - 23 2.1.3. Third PCR Step........................................................................................ - 23 2.2. Biosensor Plasmids and Strains ....................................................................... - 23 2.3. Compound Response Assays ........................................................................... - 25 2.3.1. Lead ......................................................................................................... - 25 2.3.2. Salicylate ................................................................................................. - 28 2.3.3. Selectivity Tests....................................................................................... - 29 2.4. Reverse-Transcription quantitative PCR ......................................................... - 32 2.5. Protein Electrophoresis and Western Blotting ................................................ - 36 2.6. Fluorescence Microscopy ................................................................................ - 37 3. CONCLUSIONS ..................................................................................................... - 39 4. REFERENCES ........................................................................................................ - 40 -. i.

(3) TABLE OF FIGURES Figure 1 (A) Agarose gel analysis of PCR products generated during first PCR step.- 22 Figure 2 Single-labeled biosensor plasmids. ................................................................... - 24 Figure 3 Dual-labeled biosensor plasmids.. .................................................................... - 25 Figure 4 E.coli PG biosensor strain response to several lead concentrations.................. - 26 Figure 5 Calibration curve approximations in response to lead concentrations over time. .. - 27 Figure 6 E.coli SG biosensor strain response to several salicylate concentrations. ........ - 28 Figure 7 Calibration curve approximations in response to salicylate concentrations over time. ................................................................................................................................. - 29 Figure 8 Selective response of E.coli PG biosensor to some heavy metals. ................... - 30 Figure 9 Selective response of E.coli SG biosensor to some PAHs. ............................... - 30 Figure 10 E.coli SG biosensor strain response to 1000 ppm of Toluene and Xylene. .... - 31 Figure 11 Approach followed to obtain recDNA targets for RT-qPCR experiments. .... - 32 Figure 12 Amplification chart for GFP standard curve construction. ............................. - 33 Figure 13 Standard curve with serial dilutions from 107 molecules mL-1 down to 102 molecules mL-1. ............................................................................................................... - 33 Figure 14 Melting curve analysis at the end of the One-Step RT-qPCR standardization. ... - 34 Figure 15 Change in level of GFP expression evaluated by RT-qPCR. ......................... - 35 Figure 16 TSDS-PAGE of lead biosensor at different induction concentrations. ........... - 36 Figure 17 TSDS-PAGE of GFP induction assays. .......................................................... - 37 Figure 18 Epifluorescence microscopy of lead biosensor 3 hours after induction at 25 M. ... - 38 Figure 19 Epifluorescence microscopy of lead biosensor 3 hours after induction at 200 M. .. - 38 -. ii.

(4) TABLE OF TABLES Table 1 Strains and plasmids ........................................................................................... - 11 Table 2 Primers used in this study. .................................................................................. - 14 Table 3 Lengths of RT-qPCR amplicons. ....................................................................... - 19 Table 4 Generation of recDNA targets to calibration curve............................................ - 20 -. iii.

(5) ABSTRACT Several transcriptional fusions were constructed by gene splicing by overlap extension PCR. The genetic circuits integrate the sensing system for lead and salicylate with four (4) different fluorescent proteins. Afterward, transcriptional fusions were cloned into pCR2.1TOPO to obtain a total of six (6) single-labeled biosensors. In order to obtain a dual-labeled biosensor capable of determine salicylate and lead concentration in the environment, some single-labeled biosensors were rearranged to give finally two (2) different dual-labeled biosensors. The bioreporter strains were tested against analog compounds with the aim of determine probable cross-reactivity. RNA isolations were done from several induction experiments and a One-Step Reverse Transcription quantitative PCR procedure was employed to analyze gene expression at mRNA level. Finally, inmunoblotting against GFP was done to visualize the protein and to correlate qualitatively fluorescence intensity with protein quantity. Epifluorescence studies directed to GFP revealed and verify bacterial biosensors activity.. -1-.

(6) INTRODUCTION In environmental microbiology field is of great importance to have tools that offer basic information of the environment that are being studying. Particularly, in bioremediation processes it has been of great interest to know the decontamination progress of some compounds, mainly Polycyclic Aromatic Hydrocarbons and heavy metals due to their big impact in living systems, mostly to the humans. Two general approximations to monitor chemical compounds in the environment exist. The first are physicochemical analysis conventional techniques that allow a precise and sensible determination. The disadvantage of this is that required complex and expensive equipment and specialized laboratories (Belkin 2003) and in situ measurement cannot be done, generating real information loss or modification due to the sample transport to the laboratory. Additionally,. physicochemical. techniques. failed. determining. contaminant. bioavailability and toxic effects in living systems. The second are different biological systems such as living organisms e.g. fish toxicity tests, subcellular components or enzymes that measure the chemic effect. These tests sense the direct effect of the desire compound instead of identify it. Microorganisms, mainly bacteria are a feasible option for contaminant monitor in environment because of their big population size, fast growth rate, cheap production costs and easy conservation (Belkin 2003). Having into a count the last, the molecular tools that have arisen thanks to the biotechnology improvement have helped to the development of genetically engineered microorganisms (GEM) for different purposes. Some important purposes are the improvement of some GEM to allow the biotechnological processes optimization and the synthetic biology that consist in modify living systems that posses exogenous genetic material to obtain a desired answer. The use of modified bacterial cells by gene fusions of degradation regulator systems for environmental interest compounds with fluorescent or luminescent reporter systems, whose regulation depend of the activation of the firsts have been proposed.. -2-.

(7) Bacterial bioreporter cells or bacterial biosensors defined as an artificial instrument that uses some biological system or one of its components to measure an interest compound in a desired environment are a biotechnology tool that allows determining concentrations and contaminant degradation levels during bioremediation processes. After bioaugmentation in those processes it can be propose a stable biosensor vector construction in Escherichia coli with multiple promoter-gfp gene fusions to different concern pollutant such as Polycyclic aromatic hydrocarbons and heavy metals. It is common that any measurement instrument have a noise or allowable error in its capacity to detect the interest compound. The last can be due to the cross answer with another chemically or structurally similar component to the component that is recognize by the biosensor. The first design biosensors were catalytic systems that integrated enzymes, cellular organelles, tissue or whole microorganisms (cells) that converted the biological answer to a digital electronic signal. (Turner 2000). The first concept of a biosensor was reported in 1960 and was constitute by immobilized enzymes in oxygen electrodes (Sharpe 2003). Electrochemical, optic and thermometric transductors were the principal systems used, later affinity biosensors were developed, which had an advantage because offered real time information about the antigen-antibody, cellular receptor-ligand and DNA or RNA binding with their nucleic acid complementary sequences. Biosensors have been employed to measure blood glucose content, to detect contaminants and pesticides, to monitor food pathogens or to detect biological weapons threatens (Turner 2000). Technically, a complete biosensor integrate a biological component with an electronic transductor, converting the biochemical signal in an electric quantifiable answer (D'Souza 2001). The biological component used three basic mechanisms: biocatalysis, bioaffinity or microbial cells based systems (Sharpe 2003). Biosensors that have showed satisfactory results have been based on specific interactions between enzymes and their respective substrates, the recognition between antibodies and antigens and the accessibility of a specific target molecule to its receptor (Belkin 2003).. -3-.

(8) Microorganisms have an adaptation capacity to adverse environment conditions and the ability to degrade different molecules. Bacterial cells present a wide number of advantages as a compound measurer when a biosensor is to be developed. Bacteria are ubiquitous organisms able to metabolize a broad range of chemical compounds and its genetic manipulation is relative easy e.g. mutation or recombinant DNA (D'Souza 2001). Nevertheless, the inconvenient is that large experiment periods are required. GEMs have been useful to develop biosensors that response to a specific contaminant but less have been done in the development of biosensors that involve a wide variety of bioremediation metabolic routes, the last are being developed (Sharpe 2003). Commonly reporter genes are those that codify for Green Fluorescent Protein (GFP), bacterial luciferase (lux), firefly luciferase, -galactosidase, -glucoronidase, catechol 2,3-dioxigenase and nucleation protein (InaZ) each with different advantages and disadvantages (Daunert et al. 2000; Loper and Lindow 2001; Leveau and Lindow 2002). GFP is the most known due to its easy detection by epifluorescence and confocal microscopy in individual cells and the possibility to carry out a quantitative analysis by a flow cytometry in singles cells. !-galactosidase activity can also be measure in single cells using chromogenic and fluorogenic substrates or by immunofluorescence. Luciferase has been detected in single cells, but resolution is not good, reason for the limiting use of this protein in reporter bacteria (Leveau and Lindow 2002). Although gfp and lux genes are the most used reported systems, GFP has some advantages like the possibility to be measured in single cells as described earlier. In addition, this system does not require an exogenous substrate and therefore it is not influenced by the kinetic of the cellular substrate incorporation. The disadvantage of GFP is that this is less sensible than lux in experiments where the efficiency have been compared, mostly with naphthalene (Kohlmeier et al. 2007). In the environment is not only important the specific cell biosensor answer to a contaminant, but also to emphasize and to find the sum of the negative impacts that a contaminated sample has on living systems. The last can be done by toxicity tests,. -4-.

(9) which objective is to know how much toxic the sample is and not the sample contaminant composition. For that reason, a biosensor should give the information to measure the contaminant impact that is known as the bioavailability. Belkin has categorized these detection systems in two groups, light off and light on test. The light off are microbial tests, which are based on the luminescence emission decrease measurement by the luminescent bacteria Vibrio fisheri or other microorganism that have the genetic luminescent element, when those are expose to different sample concentrations in short periods (Belkin 2003). Best approximations of that kind of tests are the light on that are based on the molecular fusions of a reporter system with specific promoters selected from different regulons related with environmental stress. Assuming that no cell reporter will be able to include all the possible potential factors of cellular stress, it has been raised that a great repertoire of these should be used (Belkin et al. 1997). In diverse studies where repertoires of bioreporter cells are used, the reporter system selected has been the microbial bioluminescence (luxCDABE) (Belkin 2003). The genetically modified microorganisms used like biosensors are typically produced with a constructed plasmid in which the genes that codify for some reporter protein are under the control of a promoter who recognizes the compound of interest. In this way, and following the reasoning of lights on systems, it is necessary to have information of the degradation, resistance, or tolerance routes for the different polluting agents present in the environment. In view of the great diversity of microorganisms, and the numerous metabolic routes that evolutionarily can diverge for a same contaminant, it is fundamental to have information for at least one of the interest bacteria. Reference strains have arisen for particular contaminants, depending on the interest or the localization of the genetic material that codify for the information. Ralstonia metallidurans CH34 is a heavy metal reference strain, isolated from zinc factory sediments. It was found that was highly resistant to Zn(II), Cd(II), Co(II), Ni(II), Cu(II), Cr(VI), Hg(II), y Pb(II). The resistance information is codify in at least seven determinant located in some of the endogenous megaplasmids pMOL28 y pMOL30. -5-.

(10) (Collard et al. 1994; Taghavi et al. 1997; Taghavi et al. 1997; Borremans et al. 2001). Pseudomonas putida G7 is the reference strain for polycyclic aromatic hydrocarbons such as naphthalene. Information is also codify by a widely characterized plasmid called NAH7, that consist of two operons for salicylate and naphthalene degradation, both of them regulated by a single regulator protein NahR (Connors and Barnsley 1982; Yen and Gunsalus 1982; Yen and Gunsalus 1985; Schell 1986; You et al. 1988; Tsuda and Iino 1990; Sota et al. 2006). Once the information of the resistance and/or degradation routes is available, the next step is to look for the interest promoter region for the desired compound and make the genetic fusion with the desired reporter gene or genes system, which finally will be regulated by the action of the effector molecule on the chosen promoter. This can be finally established at the plasmidic level and conserved intracellularly assuring the presence of an appropriate replicon with respect to the biosensor cell or to try to induce the integration of the developed construct to the chromosome by recombination. D´Souza y Lei et al., made detailed revisions of the applications and different uses of the constructed biosensor cells. (D'Souza 2001; Lei et al. 2006). The development of gene fusions is a common practice in molecular biology. These genetic elements have been used for many purposes that included the study of gene expression (Forsberg et al. 1994), as well as testing novel promoters (Bai et al. 2007), generate hybrid proteins (Sun et al. 2008), and for the development of genetically engineered whole-cell biosensors (Tamagnini et al. 2008). It is common to use progressive enzymatic digestions to join two or more DNA fragments; however, this procedure is time consuming and requires numerous extraction and transformation steps. In addition, in the construction of genetically engineered whole-cell biosensors, the regulatory systems can differ in their range size considerably. For example, the fragments including promoter region and transcriptional regulatory gene of lead determinant (pMOL30, Ralstonia metallidurans CH34) (Borremans et al. 2001), as well as the promoter of the lower pathway operon of naphthalene determinant (NAH7 plasmid, Pseudomonas putida G7) (Sota et al. 2006), are approximately of 600 bp and. -6-.

(11) 1300 bp, respectively. Furthermore, if it is considered that regulatory systems must be joined with a reporter system, which range in size around 800 bp, any transcriptional fusion generated would be of 1800 bp approximately. In an attempt to overcome these shortcomings, it could be possible to implement a modified three-steps SOE-PCR protocol, which will be fast, cost effective, and even suitable for generate genetic fusions of up to 2300 bp. SOE-PCR is an alternative protocol to construct genetic fusions, which was initially described by Higuchi et al. (Higuchi et al. 1988), and has been modified to “splice” together two desired DNA fragments in successive PCR steps. Basically, the technique consists in the generation of two independent DNA fragments by conventional PCR, both of which share a complementary region introduced by the modified primer pair used. Then, both products are mixed together and the complementary regions serve as primers, annealing and generating free 3’-ends which can be extended by a DNA polymerase. Finally, with the use of external primers, the recombinant DNA molecule is obtained at high concentrations. The efficient construction of genetic fusions by SOE-PCR might be limited by the final length of the product, and it is frequent to find studies on which the SOE-PCR protocol used to generate recombinant molecules does not achieve fusions of more than 1500 bp (Jones and Barnard 2005). When GFP is used as the reporter gene, gene expression experiments are important for two reasons. The first is to establish if a correlation between fluorescence, measured as relative fluorescence units (RFU), and protein synthesis, measured as mRNA transcripts exist. The second is to find out if basal gene expression occurs, which is referring to the expression when no contaminant is present. Different methods have been used for quantification of transcription such as western blot, northern blot, in situ hybridization, RNase protection assays and Real time PCR (RT-qPCR) (Bustin 2000). Western blot or inmunoblot is a gene expression methodology used to detect specific proteins in a given sample. First a protein separation by gel electrophoresis of native or denatured proteins is done. Then the proteins are transferred to a nitrocellulose or PVDF membrane, where. -7-.

(12) they are detected using antibodies specific to the target protein or an epitope (Renart 1979; Towbin 1979). A more recent technology is RT-qPCR, a molecular Biology methodology that can be implemented to study the differential genes expression from his lowest level that is the produced mRNA from the gene transcription. The main advantage of the technique is that it allows to quantificate the expression levels of the gene or genes of interest, since it is possible to determine initial mRNA levels present in the samples. First that all, it is necessary to extract total RNA and then it is reverse transcribed into its DNA complement (cDNA) by a RT-PCR (Reverse transcription PCR), this cDNA is what the RT-qPCR quantify by the detection of fluorescence as it accumulates in the reaction. There are three ways to generate fluorescence (Casas 2007): (i) TaqMan® Probes, (ii) Molecular Beacons, y (iii) SYBR Green I®. TaqMan® Probes were proposed by Livak et al (Livak et al. 1995) and these own a similar structure than the reporter and quencher of the Molecular Beacons. In this case a 5´exonucelase activity Taq polymerase hydrolyzes the probes that are constantly hybridized with the target DNA. This probes are designed to have around 20-30 nt, a fluorophore covalently attached to the 5’-end that is going to be hydrolyzed, a quencher molecule in the 3´-end that is phosphorylated to avoid polymerase extension and the capacity to anneal within a DNA region. As the Taq polymerase reaches the probe 5'-end, degradation of the probe releases the fluorophore from it and breaks the close proximity to the quencher, relieving the quenching effect and allowing fluorescence, which is detected by the thermal cycler used (Casas 2007). Molecular Beacons were first reported by Tyagi and Kramer (Tyagi and Kramer 1996), those are probes that have inverted repeated sequences at the ends that allow a stable hairpin formation. A fluorophore (dye) is located at the 5´-end and the quencher signal molecule at the 3´-end. In the absence of a complimentary target sequence, the beacon remains closed and no fluorescence is detected. In the presence of the complementary target sequence the beacon unfolds and fluoresces, fluorescence is detected by the thermal cycler. The last method: SYBR Green I® was a strategy proposed by Ponchel et al (Ponchel et al. 2003). When SYBR Green I® (dye) binds to. -8-.

(13) double-stranded DNA (dsDNA) emits fluorescence. As the RT-qPCR generates more dsDNA that bind dye allow to determine initial cDNA levels. Quantification in RT-qPCR can be carried out by to methods, relative or absolute. Relative quantification is easier because a calibration curve is not needed. Instead of it, it is based on internal controls or housekeeping genes, that are constitutively expressed genes at a relatively constant level whose expression is unaffected by experimental conditions. These genes have been used for studies in eukaryotes (Thellin et al. 1999; Vandesompele et al. 2002; Radonic et al. 2004; Thorrez et al. 2008), but it is still not clear what gene to use when working with bacteria. Nevertheless, there are diverse studies where the 16S rRNA gen is used in a generalized form (Lee et al. 2007; Tasara and Stephan 2007) even though it has been found that this gene can display some regulation differences in its expression under different conditions (Vandecasteele et al. 2001). Although relative quantification is a good method for gene expression levels studies, absolute quantification is more reproducible, specific and sensitive. The complexity of this method is that it requires a calibration curve to calculate the number of transcripts in the sample. It is based on standards that can be known DNA molecules concentrations. One reproducible curve consists in recombinant plasmid DNA (recDNA) as the standard due to its stability with respect to RNA, the appropriate template length and that is not necessary to synthesize, purify, clone, transform, prepare the plasmids and determine the exact standard concentration (Bustin 2000; Pfaffl 2002; Nolan 2006). In environmental microbiology RT-qPCR has been used to study resistance or degradation determinants against diverse contaminants (Saleh et al. 2005; Nyyssönen et al. 2006; Park and Crowley 2006). In biosensor studies, when cells are exposed to adverse conditions the study of the reporter genes differential expression by RT-qPCR is wide appropriate because allows to discriminate and to compare altogether fluorescence common tests with those of reporter gene expression; in this way is easier to obtain a clear analysis of the bioreporter system answer. In systems against Pb it. -9-.

(14) would be possible to determine more accurately the activation or cross answer with other heavy metal e.g. Zn (Chakraborty et al. 2008) and be able to investigate the real effect that these different effectors have on the sensor elements of the bacterial biosensors. The aim of this study was to establish the relation between fluorescence measures, transcripts copy number per cell and protein detection by inmunoblotting of the biosensors strains constructed. Also, it was constructed a pool of biosensor strains integrating several fluorescent reporter systems with sensing system for lead and salicylate. A proposal of a dual-labeled biosensor was made but those strains must be further analyzed.. - 10 -.

(15) 1. MATERIALS AND METHODS 1.1.. Reagents The lead chloride, cadmium chloride, zinc chloride, copper sulfate, nickel sulfate, mercury chloride, cobalt chloride and silver nitrate were acquired from Centro de Investigaciones Microbiológicas, CIMIC, Universidad de los Andes. Sodium salicylate were synthetically prepared and donated by Departamento de Química, Universidad de los Andes.. 1.2.. Bacterial Strains, Plasmids and Growth Conditions The bacteria strains and plasmids used in this study are summarized in Table 1. Ralstonia metallidurans CH34 and Pseudomonas putida G7 harbor the megaplasmids. pMOL30 and NAH7 which carry resistance determinants to Pb2+ and salicylate, respectively (Borremans et al. 2001; Sota et al. 2006), were grown in Luria-Bertani (LB: 10 g l-1 tryptone, 5 g l-1 yeast extract, 10 g l-1 of NaCl, pH 7.0-7.2) broth at 30ºC with shaking at 150 rpm. Unless otherwise stated, Escherichia coli DH5" and TOP10 cells harboring the plasmids were cultured in LB broth supplemented with either kanamycin (Km) (50 g ml-1) or ampicillin (Amp) (100 g ml-1) at 37ºC with shaking at 220 rpm. Table 1 Strains and plasmids Strain or plasmid Description. Reference or source. Plasmids: pECFP pDsRed2 pPROBE-NT. Ampr, ECFP. Clontech, donated by Juan Ayala. r. Amp , DsRed2. Clontech, donated by Juan Ayala. r. Km , GFP. Donated by Steven Lindow (Miller et al. 2000). r. r. pEYFP-N1. Km , Neo , EYFP. Clontech, donated by Juan Ayala. pPbCFP-s. Single-labeled biosensor plasmid. This study. r. r. Amp , Km , pbrR-pbrA’::ecfp pPbDR2-as. Single-labeled biosensor plasmid Ampr, Kmr, pbrR-pbrA’::dsred2. - 11 -. This study.

(16) pPbGFPT1-s. Single-labeled biosensor plasmid r. This study. r. Amp , Km , pbrR-pbrA’::egfp-T1 pPbYFP-as. Single-labeled biosensor plasmid r. This study. r. Amp , Km , pbrR-pbrA’::eyfp pSalGFPT1-s. Single-labeled biosensor plasmid r. This study. r. Amp , Km , nahR-nahG’::egfp-T1 pSalYFP-s. Single-labeled biosensor plasmid r. This study. r. Amp , Km , nahR-nahG’::eyfp pCext. CFP-recDNA plasmid r. This study. r. Amp , Km , #cfp pDext. DsRed2-recDNA plasmid r. This study. r. Amp , Km , #dsred2 pGext. This study. GFP-recDNA plasmid r. r. Amp , Km , #egfp pYext. YFP-recDNA plasmid r. This study. r. Amp , Km , #eyfp pPbDR2-SalGFPT1. Dual-labeled biosensor plasmid. This study. Ampr, Kmr, pbrR-pbrA’::dsred2, nahRnahG’::egfp-T1 pPbDR2-SalYFP. Dual-labeled biosensor plasmid r. This study. r. Amp , Km , pbrR-pbrA’::dsred2, nahRnahG’::eyfp Strains: E. coli DH5". F- $80lacZ#M15 #(lacZYA-argF)U169 recA1 endA1. hsdR17(rk-,. (Hanahan 1983). +. mk ) phoA supE44. thi-1 gyrA96 relA1 tonA E. coli TOP10. F- $80lacZ#M15 recA1 endA1 mcrA #(mrr-. Invitrogen. hsdRMS-mcrBC) #lacX74 araD139 #(araleu)7697 galU galK rpsL (StrR) nupG R.metallidurans CH34. Multiple metal resistant harboring pMOL30. Donated by Max Mergeay. plasmid P.putida G7. Utilizes naphthalene and harbors NAH7. Donated by Gary Sayler. plasmid E. coli PC. Lead biosensor strain. This study. DH5" containing pPbCFP-s plasmid. - 12 -.

(17) E. coli PD. Lead biosensor strain. This study. DH5" containing pPbDR2-as plasmid E. coli PG. Lead biosensor strain. This study. DH5" containing pPbGFPT1-s plasmid E. coli PY. Lead biosensor strain. This study. DH5" containing pPbYFP-as plasmid E. coli SG. Salicylate biosensor strain. This study. DH5" containing pSalGFPT1-s plasmid E. coli SY. Salicylate biosensor strain. This study. DH5" containing pSalYFP-s plasmid E. coli C. CFP recDNA strain. This study. TOP10 containing pCext plasmid E. coli D. DsRed2 recDNA strain. This study. TOP10 containing pDext plasmid E. coli G. GFP recDNA strain. This study. TOP10 containing pGext plasmid E. coli Y. GFP recDNA strain. This study. TOP10 containing pYext plasmid E.coli PDSG. Lead and salicylate biosensor strain. This study. TOP10 containing pPbDR2-SalGFPT1 plasmid E.coli PDSY. Lead and salicylate biosensor strain. This study. TOP10 containing pPbDR2-SalYFP plasmid. 1.3.. Gene-Splicing by Overlap Extension PCR It was implemented a modified three-steps gene-splicing by overlap extension PCR (SOE-PCR) protocol (Horton et al. 1989). Several transcriptional fusions integrating the fragment that comprises the promoter region and the transcriptional activator gene of the lead determinant (R. metallidurans CH34, pMOL30 plasmid) and salicylate. pathway (P. putida G7, NAH7 plasmid), along with the genes coding for fluorescent proteins CFP (pECFP), DsRed2 (pDsRed2), GFP (pPROBE-NT) and YFP (pEYFP-N1) were constructed. All stages of the three-step SOE-PCR protocol were performed in an iCycler Thermal Cycler (BioRad).. - 13 -.

(18) 1.3.1. First PCR step For the first PCR step, PCR reactions were carried out in a 25 l final volume with 1.5 l of the bacteria strains cultivated in LB broth used as DNA template, 1.5 U of Taq polymerase (Invitrogen), 1X reaction buffer, 200 M of each dNTP, and 5 pmol of appropriated primers (Table 2). The PCR conditions for all first PCR step reactions had initial denaturation temperature at 94ºC for 5 min, followed by 30 cycles of denaturation at 95ºC for 1 min; annealing at indicated temperature (Table 2) for 1 min; extension at 72ºC for 1.5 min, and finally an extension step at 72ºC for 10 min. PCRamplified DNA products were purified with a Wizard SV Gel and PCR Clean-Up System (Promega) and concentrations were assessed spectrophotometrically by NanoDrop (Rocklandm DE). The purified DNA fragments were also confirmed by sequencing with BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) using the primers listed in Table 2. Table 2 Primers used in this study. Underlined sequences correspond to complementary regions between the primers. Boldface sequences correspond to R.E sites incorporated (BamHI, PstI, HindIII). Primers Sequence (5’ – 3’) Ta (ºC) M13 forward. GTAAAACGACGGCCAG. 52.4. M13 reverse. CAGGAAACAGCTATGAC. 52.4. nah_fwd_out. GGCAAGCTTGGGCTCGTTCACTGCTAACT. 57.7. nah_BPH_rev. CTGGATCCCTGCAGAAGCTTAGCTCGTGATGGCTTTATTGATG. 57.7. pbr_fwd_out. GCGGCCGCCCAAATGCAGGAGACGAAGTA. 57.7. pbr_BPH_rev. CTGGATCCCTGCAGAAGCTTAGGGCAACCCCTTGTGTGTATTC. 57.7. cfp_HPB_fwd. CTAAGCTTCTGCAGGGATCCAGTGCAGGTCGACTCTAGAGGAT. 55.7. cfp_rev_out. ACACCAGACAAGTTGGTAATGG. 55.7. dsred2_HPB_fwd. CTAAGCTTCTGCAGGGATCCAGGACCATGATTACGCCAAGC. 55.7. dsred2_rev_out. GCTCAGTTGGAATTCTAGAGTCG. 55.7. gfpT1_HPB_fwd. CTAAGCTTCTGCAGGGATCCAGCGAGCTCGAATTCCCTAACT. 54.7. gfpT1_rev_out. CCAATTCCTGGCAGTTTATG. 54.7. - 14 -.

(19) yfp_HPB_fwd. CTAAGCTTCTGCAGGGATCCAGGCTCAAGCTTCGAATTCTGC. 59.1. yfp_rev_out. CGCTTTACTTGTACAGCTCGTC. 59.1. cfp_fwd_ext. CCGACCACATGAAGCAGC. 56.1. cfp_rev_ext. CTCCAGCAGGACCATGTGAT. 56.1. dsred_fwd_ext. CCCGTGATGCAGAAGAAGAC. 56.1. dsred_rev_ext. CCGCTACAGGAACAGGTGG. 56.1. gfp_fwd_ext. AGGTGATACCCTTGTTAATAGA. 54.0. gfp_rev_ext. CAAACTCAAGAAGGACCATG. 54.0. yfp_fwd_ext. CAGGAGCGCACCATCTTC. 58.5. yfp_rev_ext. TCCAGCAGGACCATGTGAT. 58.5. cyfp_fwd_qrt. AAGGAGGACGGCAACATC. 58.1. cfp_rev_qrt. TTGTGGCGGATCTTGAAG. 58.4. dsred_fwd_qrt. CCTGGTGGAGTTCAAGTC. 59.7. dsred_rev_qrt. GTGTAGTCCTCGTTGTGG. 59.1. gfp_fwd_qrt. GGAAGCGTTCAACTAGCAGAC. 60.5. gfp_rev_qrt. CGAAAGGGCAGATTGTGTGG. 61.2. yfp_rev_qrt. GTGGCGGATCTTGAAGTTC. 58.9. 1.3.2. Second PCR step Equimolar amounts of appropriated fragments were then mixed in the second PCR step (about 80 fmol each), with reactions performed in a final volume of 15 l with 1.0 U of Taq polymerase (Invitrogen), 1X reaction buffer, and 200 M of each dNTP. The PCR program consisted of 10 repetitive cycles with a denaturation step at 95ºC for 1 min, an annealing step for 1.5 min at 57ºC and an elongation step for 2 min at 72ºC. A final elongation step was included at 72ºC for 15 min.. - 15 -.

(20) 1.3.3. Third PCR Step The fusion products were subsequently amplified in a third PCR step reaction in a final volume of 50. l with 5. l of previous step used as DNA template, 2 U of Taq. polymerase (Invitrogen), 1x reaction buffer, 200 M of each dNTP, and 2.5 pmol of appropriated external primers (Table 2). The PCR conditions were initial denaturation at 95ºC for 5 min, followed by 30 cycles of denaturation at 95ºC for 1 min; annealing at indicated temperature to external primers (Table 1) for 1 min; extension at 72ºC for 1.5 min, and finally an extension step at 72ºC for 15 min. The transcriptional fusions were all visualized on agarose gel and stained with SYBR Green I (Invitrogen). 1.4.. Construction of the Biosensor Strains Cloning, ligation, and transformation were carried out using standard techniques (Sambrook and Russell 2001). Clones were selected by blue/white screening at 37ºC on LB agar plates containing 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal) and supplemented with either Km (50 g ml-1) or Amp (100 g ml-1). All plasmids. obtained were isolated and its structure confirmed by restriction enzyme digestion and partial sequencing which revealed identity with the original sequences. 1.4.1. Single-labeled The recombinant DNA fragments obtained at the end of the third SOE-PCR step procedure were purified with a QIAquick Gel Extraction Kit (QIAGEN) and cloned into pCR2.1-TOPO vector (Invitrogen). To achieve an efficient cloning into TA system it was performed an additional adding 3’ A-overhangs step according to manufacturer guidelines. The cloning reactions were transformed by electroporation into DH5". A total of six biosensor strains were obtained (Table 1): (i) E.coli PC, (ii) E.coli PD, (iii) E.coli PG, (iv) E.coli PY, (v) E.coli SG, and (vi) E.coli SY. 1.4.2. Dual-labeled To construct dual-labeled biosensor plasmids from E.coli PD, E.coli SG and E.coli SY strains were purified by standard alkaline lysis plasmid mini-preparation. The biosensor. - 16 -.

(21) plasmids pSalGFPT1-s and pSalYFP-s were digested with NotI (Invitrogen). Plasmids pPbDR2-SalGFPT1 and pPbDR2-SalYFP were obtained by cloning of the 1.3-kb NotI fragment containing the transcriptional fusion pbrR-pbrA’::dsred2 from pPbDR2-as into pSalGFPT1-s and pSalYFP-s linearized plasmids, respectively. The ligation reactions were then transformed into chemically competent TOP10 cells generating two dual-labeled biosensor strains (Table 1): (i) E.coli PDSG, and (ii) E.coli PDSY. 1.5.. Compound Response Assays An overnight culture of the biosensor strains were grown in LB broth with Amp (100 g ml-1) and Km (50 g ml-1). Pre-warmed LB broth supplemented with Amp (100 g ml-1) was inoculated with a 5% (v/v) volume of the overnight culture. The culture was. grown at 37ºC with shaking at 250 rpm until the OD600 reached 0.8. The cells were harvested by centrifugation at 2000g for 10 minutes and washed twice with sterile M9 minimal medium (MM) (Sambrook and Russell 2001) with 20% glucose as carbon source. Finally, OD600 were adjusted to 0.6-0.8 with sterile M9 supplemented with Amp (100 g ml-1). Next, 100-ml glass flasks () were inoculated with 10-30 ml of the cellsuspension and incubated on a shaker at 37ºC, 100 rpm. The cultures were kept in the dark to suppress photo-bleaching and preserve fluorescent-proteins. Samples were taken every hour during 5-hours at different analyte concentrations (see below). Assays were performed in triplicate. 1.5.1. Lead Biosensor strains were grown at 25 M, 50 M, 100 M, 200 M and 300 M of lead chloride to test dose-response. Basal level gene expression was evaluated at 0 M along the time (0-5 hours). 1.5.2. Salicylate Biosensor strains were grown at 200 ppm, 500 ppm, and 1000 ppm of sodium salicylate to test dose-response. Basal level gene expression was evaluated at 0 ppm along the time (0-5 hours).. - 17 -.

(22) 1.5.3. Selectivity Tests In order to determine the induction of sensing systems by compounds alike, biosensor cells were placed with equal amounts (200 M for lead; 1000 ppm for salicylate) of analog compounds as follows: (i) Cadmium, Zinc, Copper, Nickel, Mercury, Cobalt and Silver for heavy metals, (ii) Toluene and Xylene for PAHs. 1.6.. Fluorescence Assays Fluorescence measures were done for GFP induction in a VersaFluor Fluorometer (BioRad). Filters with excitation range 485-495 nm (EX 490/10, BioRad) and emission range 505-515 nm (EM 510/10, BioRad) were used, and fluorescence intensity was measured in terms of relative fluorescence unit (RFU). Cell density was measured at 600 nm in a BioMate™ 3 UV-Visible spectrophotometer (Thermo Scientific) and several estimations were done according to the formula 1 OD600 = ~2.5 x 108 cells ml-1. Fluorescence measures were normalized with reference to the OD600 value of each sample (RFU/ OD600). Assays were performed in triplicate, and the means + standard. deviations of the experiments were plotted. As internal control was used either DH5" or TOP10 strain (Table 1) in order to estimate fluorescence basal level with reference to the strain without biosensor plasmids (data not shown). 1.7.. RNA Isolation After every hour of the compound-induction experiments previously described, 1-ml samples were taken and placed immediately at -80ºC until further treatment. Afterward, samples were thawed slowly on ice and RNA was extracted with RiboPure™-Bacteria Kit (Ambion) according to the supplier’s instructions. RNA concentrations were assessed spectrophotometrically by NanoDrop (Rocklandm DE).. 1.7.1. DNase Treatment Before RT-qPCR experiments, 1. g of RNA samples were treated with DNase I. (Invitrogen) to avoid residual DNA contamination (1U/ L, 0.5 hour, room temperature). Deactivation of DNase was achieved by adding EDTA to final. - 18 -.

(23) concentration of 2.5 mM and heating at 65ºC for 10 min. Total DNA elimination was checked by standard PCR. RNA was stored at -80ºC prior to further use. 1.8.. Reverse-Transcription quantitative PCR It was performed an absolute quantitative real-time RT-PCR. Designed primers for Reverse-Transcription quantitative PCR (RT-qPCR) were 18-21 bases long (Table 2), had G/C content over 50% and a Tm of about 60ºC. The length of the PCR products ranged from 100 to 120 bp (Table 3). Beacon Designer™ software (Premier Biosoft) was used to select the primer sequences. Primers used for RT-qPCR assays were purchased from Invitrogen. Table 3 Lengths of RT-qPCR amplicons. Gene Primer pair a Amplicon length (bp) cfp. cyfp_fwd_qrt ; cfp_rev_qrt. 119. dsred2. dsred_fwd_qrt ; dsred_rev_qrt. 108. gfp. gfp_fwd_qrt ; gfp_rev_qrt. 105. yfp. cyfp_fwd_qrt ; yfp_rev_qrt. 117. a. For primer sequences see Table 2. RT-qPCR was performed with EXPRESS One-Step SYBR® GreenER™ Universal kit (Invitrogen) in a iCycler® iQ5 Real-Time detector in 96-well plates skirted PCR plates (BioRad). The composition of the PCR mix for each sample was as follows: 1 uL of DNase treated sample, 0.4 uL of reverse primer (200 nM), 0.4 uL of forward primer (200 nM), 10 uL of EXPRESS SYBR® GreenER™ qPCR SuperMix Universal (Invitrogen), 0.5 uL of EXPRESS SuperScript® Mix for One-Step SYBR® GreenER™ (Invitrogen) and 7.7 uL of DEPC-treated water, to achieve a final volume of 20 uL per reaction. Amplification program was as follows: an initial cDNA synthesis step (reverse transcription) at 55ºC (5 min), a denaturing step at 95ºC (1 min), followed by 40 cycles of 95 ºC (15 s) and 60 ºC (45 s). Melting curve analysis was done from a first step. - 19 -.

(24) starting from 55 to 95 ºC to control specificities of quantitative PCR reaction. In each run no-RT and no-template controls were included. 1.8.1. Calibration Curves and qPCR Standards Calibration curves were constructed based on recombinant DNA (recDNA) targets (qPCR standards). Briefly, PCR reactions (one by each gene) were carried out in a 25 l final volume with primers listed in Table 4. The PCR conditions had initial denaturation temperature at 94ºC for 5 min, followed by 30 cycles of denaturation at 95ºC for 1 min; annealing at indicated temperature (Table 2) for 1 min; extension at 72ºC for 1.5 min, and finally an extension step at 72ºC for 10 min. The PCR products were cloned in pCR2.1-TOPO (Invitrogen) to obtain pCext, pDext, pGext and pYext plasmids (Table 1). Integrity of cloned fragments was confirmed by sequencing with BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) using M13 primers set (Table 2). Table 4 Generation of recDNA targets to calibration curve Gene Primer pair a Amplicon length (bp). a. cfp. cfp_fwd_ext ; cfp_rev_ext. 443. dsred2. dsred_fwd_ext ; dsred_rev_ext. 282. gfp. gfp_fwd_ext ; gfp_rev_ext. 326. yfp. yfp_fwd_ext ; yfp_rev_ext. 386. For primer sequences see Table 2. The recDNA targets were produced by PCR from pCext, pDext, pGext and pYext plasmids. Then, PCR products were gel purified and quantified spectrophotometrically by NanoDrop (Rocklandm DE). Copy number of each fragment was calculated based on the length described in Table 4 with and average molecular mass of 660 Da nucleotide-pair-1. Accurate calibration aliquots from 108 molecules uL-1 down to 101 molecules uL-1 were diluted and frozen at -80ºC to avoid any type of damage.. - 20 -.

(25) 1.9.. Protein Electrophoresis and Western Blotting Tricine-SDS-PAGE of cell extracts were carried out according to Schägger (Schägger 2006). Inmunoblotting was performed as previously described (Robles et al. 2006). Primary polyclonal antibodies directed against GFP (anti-GFP, rabbit serum) were purchased from Invitrogen and used at dilutions of 1/1500.. 1.10. Fluorescence Microscopy To visualize bacteria-cells fluorescing 1-mL samples were taken from induction assays, centrifuged at 5000 x g and resuspended in 20 uL of MM. Fluorescent-cells were visualized under Zeiss Axioskop 40 fluorescence microscope (Zeiss).. - 21 -.

(26) 2. RESULTS AND DISCUSSION 2.1.. Gene-Splicing by Overlap Extension PCR. 2.1.1. First PCR step Fragments to be joined by SOE-PCR must be initially amplified by PCR. Products obtained at the end of the First PCR Step PCR were visualized on agarose gel electrophoresis stained with SYBR Green I (Invitrogen) and are shown in Figure 1A. Pbr MW(bp). Nah Size(bp). 1636. 2036 1636. 1018. 1018. CFP Size(bp). MW(bp). MW(bp). GFP Size(bp). MW(bp). YFP. Size(bp). 1636 1335. 594. 506. DsRed2 Size(bp). MW(bp). 1018. 848. 506. 1636. 1636. 1018. 1018 804. 506. MW(bp). Size(bp). 1636 981. 1018. 807. 506. 506. A. Size(bp). pbr-cfp. pbr-dsred2. pbr-gfp. pbr-yfp. MW. nah-cfp. nah-dsred2. nah-gfp. nah-yfp. Size(bp). 2161 2294 2120 2117. 1553 1420 1376 1379. B. Figure 1 (A) Agarose gel analysis of PCR products generated during first PCR step. Pbr and Nah, products corresponding to lead and naphthalene sensing systems, respectively; CFP, DsRed2, GFP, YFP, products corresponding to fluorescence proteins genes; MW, 1000 kb ladder as molecular weight marker; Size, expected length of each fragment. (B) Agarose gel analysis of PCR products after completion of third PCR step. A total of eight transcriptional fusions were generated: (i) pbr-cfp, (ii) pbr-dsred2, (iii) pbr-gfp, (iv) pbryfp, (v) nah-cfp, (vi) nah-dsred2, (vii) nah-gfp, (viii) nah-yfp; MW, 1000 kb ladder as molecular weight marker; Size, expected length of each transcriptional fusion.. - 22 -.

(27) 2.1.2. Second PCR step Although several equimolar amounts were tested for this stage, it was found that approximately 80 fmol of each fragment resulted in increased concentration of transcriptional fusions at the final stage. In a similar fashion, different sizes for complementary regions were tested (data not shown). These assays showed that 22 bp was the lower limit necessary in order to achieve the recombination of the fragments (Table 2). 2.1.3. Third PCR Step The products obtained in Second PCR Step must be amplified in a third PCR step reaction which was done in a final volume of 50 l with 5 l of previous step used as DNA template. It was also performed reactions with other volumes from second PCR step ranging from 1 l to 10 l. The results of these assays revealed that more than 5 l resulted in an increased smearing suggesting less efficiency at the final stage. On the other hand, lower volumes did not achieve better results when comparing with the volume finally used. The transcriptional fusions are shown in Figure 1B. As it was described in Materials and Methods section, it was necessary to perform an additional adding 3’ A-overhangs stage. This step could have been necessary due to the gel extraction step prior to cloning, on which 3’ A-overhangs of transcriptional fusions might have been lost. After cloning, 10-20 colonies were amplified with M13 primers (Table 2) and the products were then sequenced. Data showed that it has efficiently obtained all transcriptional fusions, in spite of the naphthalene fusions had shown less visual integrity on agarose gel electrophoresis in comparison with lead fusion (Figure 1B). 2.2.. Biosensor Plasmids and Strains Although eight transcriptional fusions were constructed by SOE-PCR (Figure 1), only six (6) single-labeled biosensor strains were obtained and confirmed by sequencing (Table 1). It may be possible that 3’ A-overhangs of transcriptional fusions had been. - 23 -.

(28) lost previously to cloning and that polyadenylation step did not recover such state. Single-labeled and dual-labeled biosensor plasmids are shown in Figure 2, and Figure 3, respectively.. Figure 2 Single-labeled biosensor plasmids. (A) Lead sensing system coupled with CFP, DsRed2, GFP and YFP reporter systems. (B) Salicylate sensing system coupled with GFP and YFP reporter systems.. - 24 -.

(29) Figure 3 Dual-labeled biosensor plasmids. Lead transcriptional fusion (DsRed2 reporter system) spliced with salicylate fusions (GFP and YFP reporter systems).. Plasmids shown in Figure 2 and Figure 3 were cloned in E.coli DH5" strain and several biosensor strains were obtained (Table 1). A total of eight whole-cell biosensor strains were obtained. 2.3.. Compound Response Assays Each GFP experiment performed was subjected to four different approaches to study dose-response expression: 1) Fluorescence measures with a fluorometer, 2) Fluorescence Microscopy, 3) RT-qPCR and 3) Inmunoblotting. However, it could not be possible to do the same approach for CFP, DsRed2 and YFP reporter systems because both fluorometer and microscope did not have appropriate filters to measure and visualize cells. Nevertheless, the other reporter systems were analyzed by RTqPCR.. 2.3.1. Lead In Figure 4 is shown the effect of lead induction along the time (0-5 hours). It is observed that biosensor cells have a maximum fluorescence emission after 3 hours of culture approximately, after which fluorescence values remain constant. There is a. - 25 -.

(30) gradual increase in fluorescence emission which is directly related with more concentration of lead chloride in solution. As a result of this assay, it was established 3 hours of culture to do selectivity tests and to take samples for further RNA isolation. The time response obtained was shorter compared with other biosensor strains constructed and described previously (Chakraborty et al. 2008). LEAD INDUCED GFP FLUORESCENCE 3400 Basal Level. RFUs/OD600. (Relative Fluorescence Units). 2900. 25 uM 50 uM 100 uM. 2400. 150 uM 200 uM 1900. 1400. 900. 400 0. 1. 2. 3. 4. 5. Time (h). Figure 4 E.coli PG biosensor strain response to several lead concentrations. Fluorescence values are normalized with OD600 value of the sample. Basal level stand for E.coli PG strain growing in absence of lead.. It is also important to notice high level of basal induction when is compared with lowest concentration tested (25. M), which could be explained in terms of either a low. restricted promoter in the lead sensing system or a self-fluorescence of the cells. Last was tested by doing the same induction experiment with host strains used to transform biosensor plasmids, which resulted in a considerable low RFU/OD600 values compared with basal level condition (data not shown). There is a regular difference in fluorescence induction between 0 M and 100 M, but such behavior suddenly changes when biosensor is subjected to concentrations of more than 150. M. This over-. induction cannot be explained exclusively as a result of a strength induction signal. - 26 -.

(31) because, as it shall be showed later, in epifluorescent microscopy images it was clear the hard effect of high lead concentration on bacteria cell viability. Nevertheless, less viable cells (just estimated by OD600 values) plus an over induction could have given the discussed result. In an attempt to determine whether lead biosensor strain could be used to sense the heavy metal over a broad range of lead concentrations, several calibration curves were constructed with data recovered at each hour (Figure 5) to check data correlation. LEAD RESPONSE CALIBRATION CURVE 3500. RFUs/OD600. (Relative Fluorescence Units). 3000 2500 2000 1500 1000 500. Hora 1 R = 0,9238 2. Hora 2 R2 = 0,9109. Hora 3 R2 = 0,8911. Hora 4 R2 = 0,917. Hora 5 R2 = 0,9237. 0 0. 25. 50. 75. 100. 125. 150. 175. 200. Concentration (uM) Hora 1. Hora 2. Hora 3. Hora 4. Hora 5. Figure 5 Calibration curve approximations in response to lead concentrations over time.. According to correlation coefficient values (R2) it might be possible to state that at 3 hours of culture it was obtained worst correlation amongst data. Despite of it was established that an induction time of 3 hours gives a stronger fluorescence measure, when it is analyzed the system as a “device” to sense lead it is better to perform measures at the beginning (1 hour) or at the end (5 hours).. - 27 -.

(32) 2.3.2. Salicylate Because of salicylate salt was prepared at lab and it was not analytical grade, the salt could have had a vast amount of other compounds; this was proved with the following results. SALICYLATE INDUCED GFP FLUORESCENCE 1600. RFUs/OD600. (Relative Fluorescence Units). 1500 1400 1300 1200 1100 1000. Basal Level 200 ppm. 900. 500 ppm 800. 1000 ppm. 700 0. 1. 2. 3. Time (h). Figure 6 E.coli SG biosensor strain response to several salicylate concentrations. Fluorescence values are normalized with OD600 value of the sample. Basal level stand for E.coli SG strain growing in absence of salicylate.. In Figure 6 are shown fluorescence response curves to 0 ppm, 200 ppm, 500 ppm and 1000 ppm of salicylate. To prepare stock solutions, it was assumed an analytical grade for sodium salicylate salt, so that the reagent concentration might have been altered. In this experiment was not possible to evidence an equilibrium point from which fluorescence signal was kept constant. It was not assayed longer exposure time because the aim of the study was to compare expression differences when it was implemented more than one reporter system with a common sensing system. So that it could be possible that at longer exposure times to salicylate salts fluorescence emission reaches a constant value (Shetty et al. 2003). Even, when it is compared basal level with 200 ppm condition resulted in no difference along time.. - 28 -.

(33) Once again were constructed several linear-fit models to get insight into GFP induction by salicylate sensing system (Figure 7). For a second time, according to results, it is preferably to make measures after 1 hour of induction. It shows that, despite of low RFU/OD600 values, when biosensor strains are placed in contact with effectors it is just 1 hour required to acquire enough data to perform an interpolation calculus. SALICYLATE RESPONSE CALIBRATION CURVE 1600 Hora 3 R2 = 0,8531. RFUs/OD600. (Relative Fluorescence Units). 1500 1400 1300. Hora 2 R2 = 0,965. 1200 1100. Hora 1 R2 = 0,9878. 1000 900 800 0. 100. 200. 300. 400. 500. 600. 700. 800. 900. 1000. Concentration (ppm) Hora 1. Hora 2. Hora 3. Figure 7 Calibration curve approximations in response to salicylate concentrations over time.. 2.3.3. Selectivity Tests One of the most important trouble regarding whole-cell biosensors is related to crossreactivity with analogous substances. That is because sensing determinants share more than a few of pathways and even more, transcriptional regulators of promoters seem to have evolved from a common ancestor (Schell 1993; Park et al. 2002; Brown et al. 2003; Hobman et al. 2005). Biosensor strains previously used were subjected to several analog compounds to check whether these react or not in a cross mode. According to discussed earlier, samples were taken at 3 hours of culture. Even, to compare the final stage with the initial stage, 1 hour samples were also taken. Results of these assays are shown in Figure 8 and Figure 9.. - 29 -.

(34) CROSS-REACTIVITY LEAD GFP BIOSENSOR 2500 Hora 0. RFUs/OD600. (Relative Fluorescence Units). Hora 3 2000. 1500. 1000. 500. 0 DH5a. Basal Level. Pb. Cd. Zn. Cu. Ni. Hg. Co. Ag. Figure 8 Selective response of E.coli PG biosensor to some heavy metals. Measures were taken at 0-hours and 3-hours of incubation. Basal level stands for biosensor strain growing without metal. DH5": refers to host cell without biosensor plasmid.. CROSS-REACTIVITY SALICYLATE GFP BIOSENSOR 2500. (Relative Fluorescence Units). RFUs/OD600. 2000. Hora 0 Hora 3. 1500. 1000. 500. 0 DH5a. Basal Level. Salycilate. Toluene. Xylene. Figure 9 Selective response of E.coli SG biosensor to some PAHs. Measures were taken at 0-hours and 3hours of incubation. Basal level stands for biosensor strain growing without PAH. DH5": refers to host cell without biosensor plasmid.. - 30 -.

(35) In the case of lead biosensor, as it was expected fluorescence measure for lead was the highest amongst treatments. Comparing heavy metal responses with basal level treatment, those values did not exceed the last so that it is possible to hypothesize that there were not cross reactivity, although there were not differences (P>0.05) For salicylate biosensor, it was again proved that the chemical prepared was not as pure as were supposed. In this case, although salicylate treatment gave more fluorescence compared with basal level, toluene and xylene resulted in more induction. It is not concluded that this effect was due to a cross reaction between NahR transcriptional regulator and PAHs chemicals. It is hypothesized that the lack of purity of salicylate did represent a low fluorescence measure, and that toluene and xylenes did induced GFP expression but no necessary more hard than with salicylate. TOLUENE AND XYLENE INDUCED GFP FLUORESCENCE 2500. RFUs/OD600. (Relative Fluorescence Units). 2300 2100 1900. Basal Level Toluene - 1000 ppm Xylene - 1000 ppm. 1700 1500 1300 1100 900 700 0. 1. 2. 3. Time (h). Figure 10 E.coli SG biosensor strain response to 1000 ppm of Toluene and Xylene. Fluorescence values are normalized with OD600 value of the sample. Basal level stand for E.coli SG strain growing in absence of compounds.. - 31 -.

(36) However, when it is plotted the dose-response fluorescence by the other types of PAHs (Figure 10), it is found again a stable fluorescence peak at 3 hours of xylenes culture but not for toluene culture. Therefore, it is probable that salicylate biosensor strain could be used as xylenes biosensor by a cross reactivity. 2.4.. Reverse-Transcription quantitative PCR Before any calculation of copy number from RNA samples by RT-qPCR employing an absolute quantification strategy, it is indispensable to construct a well-validated, reproducible and stable calibration curve. recDNA targets produced resemble mRNA transcripts by a semi-nested PCR approach as it is shown in Figure 11.. Gene dsDNA. gfp 717 bpbp 326. recDNA Like-cDNA. #gfpgfp-recDNA. CLONING. 326 bp. #gfp-qPCR amplicon. RTRT-qPCR. 105 bp. semi-Nested PCR primers. qPCR primers. Figure 11 Approach followed to obtain recDNA targets for RT-qPCR experiments. As an example, GFP case is showed.. - 32 -.

(37) After it had been produced enough recDNA targets, it was performed RT-qPCR with triplicate and the expression patterns reported in Figure 12 y Figure 13. Triplicates were run for each sample.. Figure 12 Amplification chart for GFP standard curve construction.. Figure 13 Standard curve with serial dilutions from 107 molecules mL-1 down to 102 molecules mL-1.. - 33 -.

(38) Melting curve analysis revealed specificity of primer pairs designed and the absence of any peak that could generate signal on the real-time machine (Figure 14).. Figure 14 Melting curve analysis at the end of the One-Step RT-qPCR standardization.. As it can be seen a standard curve with an overall efficiency of 88,3 %, and R2 equal to 0,968 was satisfactory produced. It has been broadly accepted a standard curve with efficiencies values between 83 % and 105 % (personal communication). Also, according to melting curve analysis the results of gene expression were all related to mRNA copies of GFP as no other DNA product was generated during One-Step RTqPCR procedure. This was also confirmed by agarose gel electrophoresis (data not shown). Taking into account absorbance values retrieved from samples of induction assays and estimating a broad viability percentages of 100 % (0 M of lead), 25 % (50 M of lead) and 1 % (200 M), it was normalized mRNA copy data to biosensor cell as follows:. - 34 -.

(39) n. ' qPCRData mRNA _ copies x 10 cell. i. i &1. (3. n. &. n. ' AbsData. 5. 2.5 x10 cells. L. % DF %. i. i &1. n. %. Viability 100. Where terms are: qPCRDatai = molecules uL-1 of mRNA from RT-qPCR data AbsDatai = OD600 value of each sample Viability = viability percentage as stated earlier DF = dilution factor during sample processing and equal to 1.5 n=3 In Figure 16 it is showed graphically induction behavior and basal levels obtained by RT-qPCR. GFP GENE EXPRESSION BASAL AND INDUCED LEVELS 10000. mRNA copiesx10-3 / cell. 1000. 0 hour 1 hour 2 hour. 100. 3 hour. 10. 1. 0 Basal Level. 50. 200. Lead concentration (uM). Figure 15 Change in level of GFP expression evaluated by RT-qPCR. Three (3) different lead concentrations and four (4) hours of sampling.. - 35 -.

(40) At 0 hours, all cultures present the same value because biosensor cells have not been in contact with metal to turn on genetic circuit more than a simple basal level. It has an average of four (4) copies of mRNA per cell at the beginning of the experiment, but when it was analyzed gene expression at 1 hour of culture is evident a toxic effect over cells because basal level down to 1 copy per cell. However, there is a clear induction of expression at 50. M and 200. M of lead as mRNA copy number values were. considerably grater than basal level: 500-fold expression at 50 M, and nearly 10000fold at 200 M. When it is compared RFU per OD600 values with mRNA copies per cell there is not a clear relationship between them because increases in RFU data are less severe than mRNA copy number. 2.5.. Protein Electrophoresis and Western Blotting Crude extracts at 3 hours of culture were analyzed by TSDS-PAGE in order to achieve higher resolution in proteins < 50kDa, where are expected GFP (26.8) and PbrR (16.4 kDa). In Figure 16 was possible to identify transcriptional regulator PbrR when the band marked was compared with DH5" band pattern.. Figure 16 TSDS-PAGE of lead biosensor at different induction concentrations. DH5" was placed as internal control of band pattern.. - 36 -.

(41) However, there was not possibility to detect on TSDS-PAGE any suspected band related with GFP. Looking forward any way for detecting GFP, it was performed an inmunoblotting procedure with polyclonal antibodies against GFP. Once again a TSDSPAGE was done and protein samples were then blotted. Although it was used an antibody directed to GFP and purchased directly (Invitrogen), there was not a clear band apparently by the dilution used (Figure 17). It would be interesting to carry out the same blotting with a low dilution and hence try to reveal it better. Nevertheless, an apparent band which becomes stronger as concentration of lead is increased is marked. The size of such band may be a little bit lower than expected value, although the band is located on a desired range with respect to standard proteins.. Figure 17 TSDS-PAGE of GFP induction assays.. 2.6.. Fluorescence Microscopy Finally, several images were taken to visualize cells fluorescing and to partially verify the results discussed previously. Although. - 37 -.

(42) Figure 18 Epifluorescence microscopy of lead biosensor 3 hours after induction at 25 M.. Figure 19 Epifluorescence microscopy of lead biosensor 3 hours after induction at 200 M.. - 38 -.

(43) 3. CONCLUSIONS Eight different transcriptional fusions were satisfactory constructed by SOE-PCR. Using this technique is feasible to join DNA fragments with ease and avoiding restriction and cloning steps. It is a key point the complementary sequences, their sizes and GC% content in order to splice without trouble two DNA fragments. A total of eight biosensor plasmids were generated integrating a single- or dual-labeled system. Such plasmids were suitable to be cloned into E.coli DH5" and TOP10 host strains and maintained because of their replicons. It is important to take into account the possibility of transforming wild-type strains to acquire bacteria cells capable of degrading and sensing. Differential gene expression for GFP was full studied, over a range of lead concentrations and along the time. Standard curve generation by using recDNA targets was done with ease and the results showed their reproducibility. TSDS-PAGE and western blot analysis of GFP expression was performed but not confirmed. It might be possible that antibodies were not enough concentrated so that band patterns did not appear clearly. Epifluorescence microscopy showed that biosensor cells constructed responded gradually to different amounts of contaminant. This result confirmed that biosensor cells were continuously expressing GFP and that at 3 hours post-induction, a toxic effect on cells is observed.. - 39 -.

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