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GENERATION OF BANK POST-TRANSCRIPTIONAL FUSIONS OF Azotobacter vinelandii. Research Center of Microbiological Sciences ICUAP-BUAP Microbial Molecular Genetics Department. Project Director: Dr. Miguel Castañeda Lucio. Project Advisor: Mtra. Silvia Ma. Del Carmen García García. Rojas Morales Emmanuel. Abstract. The production of Alginate and PHB (Poly-β-hydroxybutyrate) are produces by more microorganisms how Azotobacter vinelandii, that is a bacterium gram-negative, pleomorphic it can be two forms, dyads or cysts. The two metabolites are of importance industry, with PHB can be made surgical suture threads, as they are biodegradable in the human body. And whit alginate is used to molding pastes can be made for your teeth because it is viscosifying agent and some cosmetics. The importance of generate a bank of genes is made a stock of information of genes that produced the two metabolites. The Azotobacter vinelandii has been described in diverse works, for its wide capacity to synthesize alginate and synthesize down quality of PHB. For this reason in this work mutants of Azotovacter vinelandii affected in the accumulation of Alginate, through insert of the transposon mini Tn5 were obtained. Keywords: PHB (Poly-β-hydroxybutyrate), pUTmini Tn 5 (Transposone), Kmr (Kanamicine resistence), Smr (Estreptemicene resistence), SPCr (Espectinemicine resistence), Triparental conjugation, E. coli DH5, plasmid R6K. alginate. Thanks to their properties, the alginate is used as a thickening agent and viscosifier. It has a wide variety of applications in industries such as food, pharmaceutical and biotechnology industries. However, the large scale production of alginate is made from extracts of wild seaweed, and is too expensive, so it is of great interest to study microorganisms such as Azotobacter vinelandii, which in future will be used on an industrial scale for production of these products. INTRODUCTION In the laboratory of molecular microbial genetics is modeled to study the bacterium Azotobacter vinelandii (A. vinelandii is a proteobacterium that belongs to the family Pseudomonaceae. It is gram-negative, pleomorphic, and can be present individually or in dyads is movable by peritrichous flagella under adverse environmental conditions undergoes a process of differentiation to form desiccation-resistant structures, which are known as cysts. BMM in the lab examines the ways in which these metabolites are produced and regulated. The production of both metabolites is controlled by a dual signaling system component called Gacs / A, which consists of a kinase histidinica (Gacs) located in the cell membrane that upon receiving the appropriate stimulus is capable of self-phosphorylated and subsequently phosphorylate a response A. vinelandii has characteristics that are under study, as the fixing of nitrogen in the presence of oxygen and the versatility of your metabolism, it is capable of producing secondary metabolites of industrial interest: the intracellular polyester poly-β-hydroxybutyrate (PHB) and extracellular polysaccharide 1 regulator (GacA), which is a protein with DNA-binding motifs which acts as a transcriptional regulator of the metabolic pathways that produce alginate and PHB, in addition to controlling certain other control systems, such as post-regulator RSMA transcriptional regulator and the small RNA RSMB. In addition to alginate and PHB, the regulatory cascade controlling other genes might be involved in the synthesis of other metabolites and physiological processes. Chapter 10 pages 278-297). Are widely used in cloning and mutagenesis experiments. It is important to create tools to facilitate the work in the laboratory, using a bank of mutants will develop a series of strategies for evaluating new phenotypes and the regulation of expression of genes related to Gacs / A-Rsm. The transposon Tn5 containing the gene for resistance to an aminoglycoside antibiotic which can be expressed in a wide variety of prokaryotes and eukaryotes. Several transposons have been integrated into plasmids (eg., The Tn1 and Tn3) or chromosomes (eg., The Tn5) (Lorenzo and Timmis 1990). Transposons are therefore for a wide variety of purposes in gene technology. By transposon mutagenesis offers a variety of advantages. Mutagenesis by transposon Transposon mutagenesis is another method for introducing mutations via insertion. Transposons are known in both prokaryotes and eukaryotes and can be inserted at arbitrary locations in the genome. (Gabriela Levitus, Chapter 4, pages 53-54). A bank of mutants can be developed by use of transposons. A transposon is a mobile genetic element that can move from a genomic location to another, thanks to the presence of short repeated sequences that flank and which is capable of replicating and inserting a copy in a new location in the genome. These encode all the enzymes necessary for insertion. Mutant phenotype can be obtained with a very low rate of reversal. The integration of a transposon causes an interruption in the transcript, so that the transposon mutagenesis presents a polar effect (Maniatis, T., EF Fritsch, and J. Sambrook. 1982). Because of this the site in which the transposon is integrated in the operon can be readily determined by an assay of the activity of an enzyme or by measuring the accumulation of an intermediate product (Augustine Lopez, Lopez-Munguia page 443). BACKGROUND Transposons They have developed a series of transposon mutagenesis to perform in the laboratory the most used are the mini-transposons as they have many advantages, are easy to use, possess variants with resistance genes. (The most cited in the literature are the mini Tn5 and Tn10 mini Lorenzo and Timmis 1990). Insertion mutagenesis with pUTmini-Tn5 The mini-transposon mini-Tn5 transposon is derived from Tn5 compound. Each of these elements lack the gene coding for a transposase gene and include, which confers resistance to an antibiotic, flanked by a sequence of 19 bp from the ends of the I and O Tn5. In our case we used the mini-Tn5 Sm containing the gene that gives resistance to Sm. (Way et al., 1984). The mini Tn5 transposons are mini-transposons that are transferred using a suicide plasmid origin of replication R6K (Lorenzo and Timmis 1990) and offer a selection of markers with various antibiotics such as Streptomycin / Spectinomycin, Kanamycin, Streptomycin, Tetracycline, Chloramphenicol . It has been determined showing a transposition frequency of 10-5 to 10-6, but may vary with different bacterial species (Michael T. Jack Parker The transmission vector, plasmid pUT (de Lorenzo et al., 1990) has a replication origin πdependent protein (derived from plasmid R6K) 2 and the transfer origin derived from plasmid autoconjugativo oriT RP4. Furthermore the plasmid pUT has the gene that encodes transposase needed for transposition of the mini-Tn5. R6K (π-dependent protein), carries the transposase gene tnp cis and whose conjugal transfer to recipient cells is mediated by RP4 mobilization functions in the donor (Michael T. Jack Parker Chapter 10 pages 278-297) The pUTmini-Tn5 is stable and capable of being mobilized, as in strain S17.1 pir , this strain produces a protein expressed π and functions for the conjugation of RP4. Mutagenesis on the plasmid is transferred by conjugation to a non-producing strain of the protein and thus can not maintain the plasmid. There are many cases of conjugative plasmids, but some of them can be transferred to recipient strains, when "living" in the donor cell with another capable of mobilizing conjugative plasmid. This is usually due to these plasmids mobilized autotransmisibles but lack the tra region but have a region called the mob (the "mobilizable"), which is recognized by Tra conjugation proteins supplied in trans by the plasmid mobilizing, although the region mobilizable plasmid mob region provides a cis (sequence oriT) and a pair of functions in trans (specific endonuclease which acts on oriT, and a helicase) (Charles Round, Guillermina Alonso September 10, 2007). The conjugants selected by antibiotic resistance encoded by the mini-transposon mutants are mini-Tn5 insertion. The advantage is the use of mini-transposon is that having no transposase encoded generate stable insertions. Also in the case of mini-Tn5 Sm has no transcription terminator sequences flanking the gene for streptomycin, which reduces polar effects. For the plasmid can be mobilized pUTmini Tn5 plasmids does so through so-called "helper plasmid" that may force him Mob + plasmid work to mobilize (Guadalupe Espin 2006). The strain of E. coli DH5 pir containing the pir gene and is designed for cloning and propagating plasmids with R6K replication origin like pUTmini Tn5. To transfer these plasmids directly from a donor strain DH5 pir to a recipient strain is necessary to carry out a triparenteral conjugation with a helper strain such as E. coli DH5a pRK2013 (Guadalupe Espin 2006). The procedure followed for the mutagenesis is that described by Lawrence et al., 1990. Mini Tn5 transposons are constructed in a manner to simplify the generation of substantial insertion mutants, fusions in vivo as reporter genes are the gusA gene or Lac Z and the introduction of foreign DNA fragments in the chromosome of a wide variety of bacteria including gramnegative enteric bacteria and soil such as Pseudomonas species. The mini-transposon consisting of genes specifying resistance to an antibiotic (kanamycin, streptomycin, chloramphenicol, tetracycline), as selection markers and a unique NotI cloning site, flanked by 19 bp repeat terminal Tn5. Other derivatives of Tn5 also contain lacZ genes. PhoA, lux AB, or xylE lacking its native promoter located after the terminal repeats in an orientation that provides the generation of gene fusions operons (Maniatis, T., EF Fritsch, and J. Sambrook. 1982). The bank will be constructed transcriptional fusions using the gusA reporter gene encoding β-glucuronidase for molecular biology, the βglucuronidase is used as a reporter gene to control gene expression in mammalian cells, plants, and microorganisms (Carlos Pena and Enrique Galindo 2001). Its monitoring is performed by using a GUS assay which allows determination of the expression of the gene in question (iván darío barrero-Farfán February 11, 2008). Transposons are located on a suicide plasmid which carries a replication origin from plasmid 3 The bank will be constructed transcriptional fusions introducing a gusA reporter gene into the NotI site of plasmid pUTmini Tn5, which will carry out the transcriptional fusion with the reporter gene under the control of a gene upstream of it. Because the reporter gene has its own sites of initiation of translation, as the reporter gene product is not regulated translationally, the amount of reporter gene product is proportional to the transcription of the gene upstream of the reporter. The translation of the mutated gene stops before the reporter gene, and translation restart at sites of translation initiation in front of the reporter gene (Primrose et al. 2001). Therefore, as a result there is the product of two genes. The truncated protein and reporter protein. OBJECTIVES GENERAL PURPOSE Build a bench transcriptional fusions. SPECIFIC OBJECTIVES 1. Establish conditions for obtaining a significant number of mutant clones enough to have represented the genome of Azotobacter vinelandii. 2. Test the conditions of a massive mutagenesis experiment. MATERIALS AND METHODS Conjugation triparenteral The method of conjugation triparenteral for transformation of strain E, Azotobacter vinelandii, using the plasmids from E. pUTmini coli Tn5 Km-resistant and Sm and the helper plasmid pRK2013 kmr. When performing the construction of genomic libraries is important to obtain sufficient numbers of clones as this will be represented in these whole genome to be studied, in this case the genome of Azotobacter vinelandii is about 5.300 Kb so to have represented all of its genome in a bank require an approximate amount of 5000-6000 colonies each with insertions in different fragments (Guadalupe Espin March 6, 2009). In practice, the size of the library can be calculated simply on the basis of the probability that a particular sequence to be represented in the library (Brown 2001). There is a formula that takes into account certain factors and results in the "number of clones" that we should consider to have represented the genome that interests us, the formula is: Strain of A. vinelandii is a mucosal morphology in the colonies formed. For conjugation is started with a culture of A. vinelandii in a 250 ml Erlenmeyer flask with 50 ml of nalidixic acid BS more liquid medium was inoculated with two roast A. vinelandii from a fresh plate, was incubated with stirring for 24 hours at 30oC. N = ln (1 - P) / ln (1 - a / b) Where N is the number of clones required, P is the probability that a particular sequence desired to be represented (typically set at 0.95 or 0.99), a is the average size of DNA fragments to be cloned, b is the genome size (expressed in the same units as a). Using this formula, we can determine the magnitude of the task and plan a strategy for cloning. To prepare a culture of strains of E. coli (pUTminiTn5 Smr and the helper pRK2013 Kmr) in 15 ml falcon tubes with 5 ml liquid LB medium plus its respective antibiotic Sm and Km, was incubated for 12 hours at 30oC. 4 The next day, the cultures obtained from E. coli were inoculated, other 5ml of LB medium without antibiotics and incubated for 3 hours under the same conditions as before. Crops are removed E. coli and A. vinelandii completed incubation times. helper pRK2013 Km and the recipient strain of Azotobacter vinelandii. During the different transformants were not obtained enough when tested under different conditions for the donor strain of E. coli containing plasmid mini Tn5 Km and the recipient strain of Azotobacter vinelandii with and without helper plasmid, with different ratios tested. Example: A. Vinelandii is planted at 11:00 am Incubate 24 hours at 32°C Remove the culture at 11:00 am Strains of E. pUT mini Tn5 coli and seeded Helper at 8:00 pm In subsequent trials continued testing conditions for the donor strain of E. coli containing plasmid mini Tn5 Sm and the recipient strain of Azotobacter vinelandii with and without the helper plasmid in different proportions. It was observed that the number of transformants was greater with the helper plasmid such that the test was continued using is. Incubate for 12 hours at 37°C Inoculate a tube with 2 ° LB without antibiotic and incubated at 37 ° C 3hr The culture of A. vinelandii, centrifuged in two tubes or Falcon sterile 1.5ml eppendorf tubes and washed as often as necessary with 10 mM MgSO4 to remove the alginate present in the cells, (recommended to be careful when discarding the supernatant, not to remove cells) to obtain the tablet. Found suitable proportions to obtain a sufficient number of transformants using donor strain of E. coli containing the plasmid miniTn5 Sm and the recipient strain of Azotobacter vinelandii and the helper plasmid in ratios 1:5.1 respectively. Colonies were chopped and replanted for conservation. Conjugation is performed using different ratios of the plasmid donor strain and helper receptor in cross plates plus antibiotic selection as appropriate. Sample dilution: No. Box 1 Plates were incubated for 48 hours at 32°C, after which time the colonies is observed with the characteristic morphology of A. vinelandii, and obtain a sufficient number of colonies to be represented the genome of A. vinelandii. Relationship 1:5:1 Proportion 25 µl de mini Tn5 Smr + 125 µl de A. Vinelandii + 25µl pRK2013 Kmr 2 miniTn5 Smr A. Vinelandii pRK2013 Kmr 1 miniTn5 Smr A. Vinelandii pRK2013 Kmr RESULTS 3 miniTn5 Smr A. Vinelandii pRK2013 Kmr We tested different proportions between the donor strain of E. coli containing the plasmid mini-Tn5 Km or Sm and the recipient strain of Azotobacter vinelandii. And moreover different proportions between the donor strain of E. coli containing the plasmid mini-Tn5 Km or Sm, the strain of E. coli containing the plasmid Conjugation plates this picture shows the arrangement of the dilutions. 5 set for the bench of mutants of Azotobacter vinelandii take time as it is a microorganism that requires a culture time greater than other microorganisms such as E . coli, and due to its characteristics such as alginate production make difficult the recovery of a sufficient number of cells to perform the tests. Several tests were required to establish the proper proportions to obtain a sufficient number of transconjugant colonies. Conjugation plates after 48 hours at 32oC. These plates were incubated for 24-48 hours at 32oC.. After obtaining the product of conjugation is collected a certain number of colonies on other boards. The composition of the medium is the same as that of conjugation. PERSPECTIVES Enter the gus gene into the NotI site of plasmid pUT mini Tn5 that will be used in obtaining banking fusions. Get banking transcriptional fusions with the conditions established in the wild strain of Azotobacter vinelandii. Chop the colonies and replanting for conservation. CONCLUSIONS Found suitable proportions to obtain a sufficient number of transformants using donor strain of E. coli containing the plasmid miniTn5 Sm and the recipient strain of Azotobacter vinelandii and the helper plasmid in ratios 1:5.1 respectively. Were grown about 150 colonies per plate observed the characteristic morphology of Azotobacter vinelandii. It stung about 1000 colonies for conservation. The length of stay was insufficient to meet all the objectives as the conditions and proportions 6 REFERENCES 1. Victor de Lorenzo, Marta Herrero, Ute Jakubzik, and Kenneth N. Timmis. 1990. Mini-TnS Transposon Derivatives for Insertion Mutagenesis, Promoter Probing, and Chromosomal Insertion of Cloned DNA in Gram-Negative Eubacteria. GBF-National Research Centre for Biotechnology, Braunschweig, Federal Republic of Germany. 2. Michael T. Madigan, John M. Martinko, Jack Parker. Biología de los microorganismos Brock 10a edición. Capítulo 10 Genética Bacteriana, Tema: Técnicas de genética bacteria in vivo. (Paginas 278-297). 3. Gabriela Levitus, Viviana Echenique, Clara Rubinstein, Esteban Hopp y Luis Mroginski. Biotecnología y mejoramiento Vegetal II. Capítulo 4, Tema: Herramientas Básicas de Ingeniería Genética, sección: Introducción del vector en la célula hospedadora. (Paginas 5354). 4. 8. Anja Knietsch, Tanja Waschkowitz, Susanne Bowien, Anke Henne and Rolf Daniel. Construction and Screening of Metagenomic Libraries Derived from Enrichment Cultures: Generation of a Gene Bank for Genes Conferring Alcohol Oxidoreductase Activity on Escherichia coli. 9. Martín Peralta-Gil, Daniel Segura, Josefina Guzmán, Luis Servín-González and Guadalupe Espín. Expression of the Azotobacter vinelandii Poly-βHydroxybutyrate Biosynthetic phbBAC Operon Is Driven by Two Overlapping Promoters and Is Dependent on the Transcriptional Activator PhbR. 10. Daniel Segura, Odon Vite, Yanet Romero, Soledad Moreno, Miguel Castañeda and Guadalupe Espín. Isolation and Characterization of Azotobacter vinelandii Mutants Impaired in Alkylresorcinol Synthesis: Alkylresorcinols Are Not Essential for Cyst Desiccation Resistance. Agustín López, Mariano García Garibay, Rodolfo Quintero Ramírez, Agustín LópezMunguía Canales. Biotecnología alimentaria. Alginatos. (Página 443). 11. Miguel Cocotl, Soledad Moreno, Josefina Guzmán, Miguel Castañeda y Guadalupe Espín. PsrA, RpoS y RsmA forman parte de una cascada de regulación que controla la síntesis de alginato y PHB en A. vinelandii. Instituto de Biotecnología, UNAM. 5. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 6. Carlos Peña y Enrique Galindo. Cambios en la morfología de Azotobacter vinelandii y su relación con la síntesis de alginato y PHB. Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México. 7. Gerardo Gaona, Cinthia Núñez, Joanna B. Goldberg, Alicia S. Linford, Rebeca Nájera, Miguel Castañeda, Josefina Guzmán, Guadalupe Espín, Gloria Soberón-Chávez. Characterization of the Azotobacter vinelandii algC gene involved in alginate and lipopolysaccharide production. 7