Printer Friendly

Evaluating alfa-1 antitrypsin properties in Saccharomyces yeast (practical study on PYNIU-AT).

INTRODUCTION

Serpins are the main parts of the protein with the different duties and turnovers which is including halter and also unhalter activities such as vocalization of lobe [15]. Serpins of clinically interesting because of the mutation which is available in their structure cause many diseases such as coagulation defects and a number of diseases also caused by structural changes and polymerized protein molecule [20, 11]. Restricting motion of human plasma proteases was determined by Fermi also Pernossi in 1984, in addition the central inhibitor clarifying for anti-proteolytic was at the beginning decomposed by Shultz in 1955, and it was named Alpha-1 Antitrypsin. Human alpha 1-antitrypsin (AAT), a serum glycoprotein, is one of the best-known models of serine protease inhibitors (serpins) superfamily [1, 17]. Alpha 1-proteinase is the main neutral serine proteinase in serum, especially Elastase, which enjoys three different additional Oligo-Saccharides, and it is 12% of its weight. Iso-electric pH of this protein varies between 4.4 and 7.4. An average amount of about 34g of this protein is synthesized by liver and secretes into serum [3, 5, 26]. In addition, AAT exists in the other types of cells, including plackets, pancreas megakaryocytes, langerhans islands [8, 14]. Its density is between 1.5 up to 3.5 gr/lit, and its plasmid half-life is between 4 to 8 days [22]. AAT forms a strong complex with proteases which is an irreversible reaction [4, 16, 23]. Ultimately, reduction of secretion results in this inhibitor shortage, and then leads to some health problems such as pulmonary emphysema [19, 28]. Giving pure AAT to these patients decreases the disease side effects, and due to its broad treatment application, protein exploitation process could be designed from human blood [24]. Hence, its recombinant production through various sources such as Eukaryotic and prokaryotic micro-organisms is important for genetic manipulation facility and mass production [29]. In this regard, since AAT is the result of mass produced Glycosylated, consistent yeast, Eukaryotic micro-organisms is preferred. After AAT expression in Saccharomyces cerevisiae yeast, expression increased through fermentor, and the properties of this protein, including molecular mass, and inhibition are measured.

Materials and Methods

The intracellular serpins:

Serpins in high Eukaryotes are devided into zgroups:

The intra cellular serpins or ovoserpins making a complete clear group and are the ancestor of extracellular serpins. The adjustment of cell behavior is a completely understandable subject under the obscurities of serpins. Megsin is a member of serpines which is sustaining the megacaryocyte of marrow. Plasminogen has an ability to restrain tumor necrosis factor which is known as an element of apoptosis and MENT is along with agglomerating cromatine. Some of the interacellarserpins or (ovoserpins) are performing the restraining roll. For example PI-6 restrains captain G but the activities of interacelluharserpins still unknownable. Any way by the exception of the ovoalbomyne which is a non-inhibitory serpins, all the interacelluralserpins including parts of knee is for hibitory activities. OVO albumen which is the main part of the egg call it as an albumen, first of all use as a sauing proteins elements. Recent studies show that ovo albumen from its structural arrangement during embryonic development the extracellular serpins. The extracellular serpins; extracellular serpins are devided in 8 group called as a/b/c/d/e/f/g/h. Among them the biggest group is a, which is contouningserpins like alfa-1-antitrypsine the members of this group are in relationship with wide range of processess which the common one is inspiring the protease. For example R A S P (calistatin) alfa-1-antitrypsine, antikimotrypsine are available in this group. Some of the non-inhibitory serpins such as CBG which are transmitting the hormones and those globolines which are adjoining to (TBG-tirocsine) and peptide hormones (the carrier of angiotansionogen, vterineserpines like UTMP (uterine milk protein) and UFAP (utera ferine associated protein) are including in extracellular serpins in b/c/e groups. Uterine serpines are extremely branching and receptacle non-inhibitory knee area which their activity still mysterious. Joining ovine UTMP to the growth factor which has surveyed indicate that this serpine is completely effective to seprate the active from pregnant uterus. In group F, PEDF (pigmenteEthelium Derived Factor) serpines is available. Which is imagine that is a not o filli factor and [[alpha].sub.2] (anti plasmine) which is known as a serpine in moray have common ancestry with mammels in group F. group d is including cofactor F (heparin). Group (g) including [C.sub.1] restrainer and in h group hsp 47 serpines are available which never have restrainer activity, and exactly they act like active pap ones in folding procelagen. Micro organisms which are used in researching for doing this research they use unidirectional bacteria and zymogenic which is indicating bellow:

1. DH5[alpha] lineage (achrichiakli) it's a bacteria lineage which is produced by stratagene company, it is used for reproduction and keeping pelasmids. Its genetic profile as follows: (F-gyr A 96 Nlae, rec AL thi-1 hsd R 17 r-k mtk)

2. Zymogenic lineage it is used for AAT recombinant protein, as long as it has shown in genetic plans, (ura3) tubidged has been activated in this zymogenic lineage. Genetic profile (MATa pep4: his 3 prb - [DELTA]1.6R could 1, his 3-20, ura 3-52)

3. Plasmid PYINU-at, vector indicates [alpha]1-AT human zymogenic one vector in dicate 2[micro] based. This image indicate that how vector Manu fractured?

Chemical materials and biologic usages in researching: subculture of bacteria LB (Luria Bertani subculture of hiquid Bacteria (LB)

Bactopepton (Dif Co) 10 g

Zymogenic extract (Dif Co) 5g

Salt (NaCl) (Merck) 10g

Distilled water to 1litre volume

Subculture of bacteria solid LB

Bacto peptone (Dif Co) 10 g

zymogenic extract (Dif Co) 5g

Salt (Merck) (NaCl) 10g

Aga, powder (Mir Midia) 15g

Distilled water (to 1 liter volume)

Put these materials into the clean flasks then add distilled water up to 1 liter volume. After that all these materials dissolve in water, strill the culture environment by auto clave.

Results and Discussion

According for table 1, SDS-PAGE gel is related to saccharomyces cerevisiae, a zymogeni fermentation which is containing AAT gen with inspiration and cycle engine is depending on oxygen from left to right zero indicate a time before on inspiration and 1 to 5 hours regularly indicate after inspiration.

[GRAPHIC 1 OMMITED]

According to these cases at the continuances of zymogene fermentation of saccharomyces cerevisiae with AAT Gen and the starting of inspiration during 3/4/5/6/7 hours after insemination with IPTG 0.4MM with cycle engine which is related to oxygen had done.

Tables 2 to 6 with graphs indicate those explanations.

[GRAPHIC 2 OMMITED]

[GRAPHIC 3 OMMITED]

[GRAPHIC 4 OMMITED]

[GRAPHIC 5 OMMITED]

[GRAPHIC 6 OMMITED]

Conclusion:

Serpin A1, also known as Alpha-1 antitrypsin (AAT), is a prototype member of the serpin super family of the serine protease inhibitors [12]. This serine protease inhibitor blocks the protease, neutrophil elastase. Alpha-1 antitrypsin is mainly produced in the liver and acts as an antiprotease [18, 13]. Its principal function is to inactivate neutrophil elastics, preventing tissue damage. Alpha1-antitrypsin, an acute phase protein and the classical neutrophil elastase inhibitor, is localized within lipid rafts in primary human monocytes in vitro [27]. It association with monocytes is inhibited by cholesterol depleting/efflux-stimulating agents (nystatin, filipin, MbetaCD (methyl-beta-cyclodextrin) and oxidized low-density lipoprotein (oxLDL) and conversely, enhanced by free cholesterol. Furthermore, SerpinA1/monocyte association per se depletes lipid raft cholesterol as characterized by the activation of extracellular signal-regulated kinase 2, formation of cytosolic lipid droplets, and a complete inhibition of oxLDL uptake by monocytes [7, 25]. Previous population studies have suggested that heterozygote status for the AAT gene (SerpinA1) is a risk factor for chronic rhinosinusitis with nasal polyposis (CRSwNP). Alpha-1 antitrypsin deficiency is a recently identified genetic disease that occurs almost as frequently as cystic fibrosis. It is caused by various mutations in the SerpinA1 gene, and has numerous clinical implications [2]. Alpha-1 antitrypsin deficiency is an inherited disease affecting the lung and liver. In the liver, alpha-1 antitrypsin deficiency may manifest as benign neonatal hepatitis syndrome; a small percentage of adults develop liver fibrosis, with progression to cirrhosis and hepatocellular carcinoma [6, 10]. It's most important physiologic functions are the protection of pulmonary tissue from aggressive proteolytic enzymes and regulation of pulmonary immune processes [21]. It is apparent that Zymogen IA is similar to hog pepsinogen in several respects. It is known that hog pepsinogen is stable in a mild alkaline medium in which hog pepsin is unstable. The observation that gastricsin and human pepsin are produced from the activation of the Zymogen IA fraction suggests two possibilities. (a) Zymogen IA is a common precursor for gastricsin and human pepsin, and (b) Zymogen IA consists of two zymogens, pepsinogen and a zymogen for gastric sin. The evidences in favor of the "common precursor" are (i) Zymogen IA appeared as a single boundary in ultracentrifugation studies and as a single band in starch gel electrophoresis; (ii) the ratio of pepsin to gastricsin varied as a function of the activating pH whereas the total amount of enzyme produced from Zymogene. Gastricsin and human pepsin were both found to be present as a zymogen in human gastric mucosa. In the attempts to purify the zymogen, a fraction (IA) has been obtained from the alkaline extract of human gastric mucosa after ammonium sulfate precipitation and diethylaminoethyl cellulose chromatography. Zymogen IA behaved as a single component in ultracentrifugation and starch gel electrophoresis. It was stable in a solution of pH 8.5. However, once acidified to pH 5 or below, it was no longer stable at pH 8.5. When acid-activated Zymogen IA was fractionated in an Amberlite IRC-50 column or by starch gel electrophoresis, gastric sin and human pepsin were obtained. The physicochemical and enzymatic properties of the enzymes obtained from the activation of Zymogen IA were identical to gastric sin and human pepsin isolated from human gastric juice. It appeared that both enzymes might be derived from this same zymogen. Single-chain pro-urokinase (pro-uPA) is present both in the medium and lysate of the A431 epidermoid carcinoma cell line. Most of the cell-associated pro-uPA is on the cell surface, as shown by indirect immunofluorescence and by surface lodination. Pro-uPA is not an integral membrane protein but is bound to a specific surface receptor that is completely saturated [9]. A mild acid treatment uncovers the surface receptors by dissociating pro-uPA. Resaturation of uncovered receptors has been studied by reincubating cells in normal medium; within 40 min, 50% of the free sites are reoccupied. Excess uPA-specific antibodies prevent rebinding of ligand to the receptors. Thus, A431 cells first secrete uPA, which then binds to the surface receptor. We propose that the synthesis of uPA and uPA receptor by the same cell may provide a pathway for the activation of the metastatic potential of malignant cells.

Article history:

Received 12 September 2013

Received in revised form 25 November 2013

Accepted 29 November 2013

Available online 16 January 2014

REFERENCES

[1] Arjmand, S., A.S. Lotfi, M. Shamsara, S.J. Mowla, 2013. Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastoris, Ejbiotechnology, 16. DOI: 10.2225.

[2] Beathy, K., J. Bieth and J. Travis, 1980. Kinetics of association of serpine proteinase with native and oxidase alpha-1-proteinase inhibitor and alpha-1-anti chymotrupsin. J. Biol. Chemist., 255: 3931-3934.

[3] BradFord, M.M., 1976. rapid and sensitive method for quantitaion of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem., 12: 248-254.

[4] Carrell, R.W., J-O. Jeppsson, C-B. Laurell, S.O. Brennan, M.C. Owen et al, 1982. Structure and variation of human [[alpha].sub.1]-antitrypsin Nature., pp: 298-34.

[5] Cox, D.W., V.D. Markovic, I.E. Teshima, 1982. Genes for immunogolobulin heavy chain and for alpha-1 antitrypsin are located to specific regions of chromosome., 14(279): 428-430.

[6] Davis, I.D., B. Bruke, D. Freese, H.I. Sharp, Y. Kim, 1992. The palnologic spectrum of the nephtopathy associated with [[alpha].sub.1]-antitrypsin deficiency. Hum. Pathol., 23(1): 57-62.

[7] Einhauer, A., M. Schuster, E. Wasser Bauer and A. Jungbauer, 2002. Expression and purification of homogenous proteins in saccharomyces cerevisiae based on ubiquitin--FLAG fusion. Protein Expression and purification, 24: 297-504.

[8] Erlanger, B.F., N. Kokowsky, W. Cohen, 1961. Preparation and properties of two new chromogenic substrate of trypsin. Arch Biochem, 95: 271-8.

[9] Hill, J., K.A. Donald, D.E. Ianand Griffiths, 1991. DMSO-enhanced whole cell yeast transformation. Nucleic Acids Research, 19(20): 5791.

[10] Hoffman, C.S. and F.A. Winston, 1987. Ten-minute DNA Preparation from yeast efficiency releases autonomouse plasmids for transformationof Escherichia Coli., Gone, 57: 267-272.

[11] Huntington, J.A., R.W. Carrell, 2001. Theserpins: nature's molecular mousetraps science prog., 84(2): 125-136.

[12] Irving, J.A., R.N. Pike, A.M. Lesk, J.C. Whisstock, 1845-1894, 2000, Phylogeny of the serpin superfamily: implication of patterns of amino acid conservation for structure and function, 10 (12).

[13] Janciauskiene, S., 1991. Conformational properties of serine proteinase inhibitors (serpins) confer multiple pathophysiological roles. Biochim. Biophys. Acta., 1535: 221-235.

[14] Kim, T.H., J.K. Jung, S.S. Kwak, S.W. Nam, M.J. Chun and Y.h. Park, 2002. Heterologous expression and secretion of sweet potato peroxidase isoenzyme A1 in recombinant saccharomyces cerevisiae. Biotechnology Letters, 24: 279-86.

[15] Lai, E.C., F.T. Kao, M.L. Law, S.L.C. Woo, 1983. Assignment of the alpha-1-antitrypsin gene and sequence-related gene to human choromosome 14 by molecular hybridization. Am. J. Hum. Genet., 35: 385-392.

[16] Lomas, D.A., A. Lourbakos, S.A. Cuming, Y.D. Belorg, 2002. Hypersentive mousetraps, [[alpha].sub.1] antitrypsin deficiency dementia. Biochem. Society Transactions., 30(2): 89-92.

[17] Majdi, S., A.S. Lotfi, S. Papi, M.F. Moghadam, Z. Joneidi, F. Memari, L. Farzampanah, M. Negahdary, 2013. Separation and Optimization of Alpha-1 Antitrypsin Properties in Saccharomyces Yeast. European Journal of Zoological Research, 2(6): 36-43.

[18] Malley, K.M.O., S.A. Nair, H. Rubin, B.S. Cooperman, 1997. The kinetic mechanism of serpin-proteinase complex formation an intermediate between the michaelis complex and the inhibition complex. J. Biol. Chem., 272(8): 5354-59.

[19] Nukiwa, T., M.L. Brantly, F. Ogushi, G.A. Fells, R.G. Crystal, 1988. Charactrization of the gene and protein of the common alpha-1-antitrypsin normal M2 allele. Am. J. Hum. Genet., 43: 322-330.

[20] Nukiwa, T., M. Brantly, F. Ogushi, G. Fells, K. Satoh, et al., 1987. Characterization of the M1 (Ala 213) type of [[alpha].sub.1]-antritrypsin a newly recognized. Common normal [[alpha].sub.1]-antitrypsin haplocyte. Biochemistry, 26: 5259-5267.

[21] Orlean, P., A. Herscovice, 1993. Glycoprotein Biosynthesis in yeast, The FASEB Journal, 7: 540-550.

[22] Picard, V., M. Dautzenberg, B. Villoutreix, G. Orliaguet, M. Alhenc-Gelas and M. Aiach, 2003. bloodjournal. Antithrombin Phe229Leu: a new homozygous variant leading to spontaneous antithrombin polymerization in vivo associated with severe childhood thrombosis., 102: 919-925.

[23] Sambrook, J., D.W. Russell, 2001. Molecular Cloning, A laboratory manual. 3rd edition, cold spring Harbor Laboratory press, New York, U.S.A.

[24] Sifers, R.N., B.B. Rogers, H.K. Hawkins, M.J. Finegold, S.L.C. Woo, 1989. Elevated synthesis of human [[alpha].sub.1]-antitrypsin hinders the secrection of murine [[alpha].sub.1]-antitrypsin from hepatocytes of transgenic mice. J. Biol. Chem., 264: 15696-700.

[25] Sveger, T., 1985. Plasma proteinase inhibitors in [[alpha].sub.1]-antitrypsin deficneint children. Pediatr Res., 19: 834-841.

[26] Travis, J., G.S. Salveken, 1983. Human plasma proteinase inhibitors. Ann. Rev. Biochem., 52: 655-709.

[27] Tuite, M.F., J.J. Clare, M.A. Romanos, Expressing cloned genes in the yeasts saccharomyces cerevisiae and pichiapastoris, pp: 61-100.

[28] Vaughan, L., M.A. Lorier, R.W. Carrell, 1982. [[alpha].sub.1]-antitrypsin microheterogencity: Isolation and physiological significance of isoforms. Biochim Biophys Acta., 701: 339-45.

[29] Wanger, S.L., A.L. Lau, D.D. Cunningham, 1989. Binding protease nexin-1 to the fibroblast surface alters its target proteinase specificity. J. Biol. Chem., 264: 611-15.

(1) Sahar Majdi, (2) Abbas Sahebghadam lotfi, (3) Maryam Ghodrati siahmazgi, (4) Reyhaneh Chelongar, (4) Raheleh Behrooznam, (4) Samira Vaziri, (5) Mehdi Manbachi, (6) Masoumeh Rajabi Bazl, (7) Masoud Negahdary

(1) Young Researchers and Elite Club, Tehran Medical Branch, Islamic Azad University, Tehran, Iran.

(2) Department of Clinical Biochemistry, TarbiatModares University, Tehran, Iran.

(3) Department of Biochemistry, Science & Research Branch, Islamic Azad University, Tehran, Iran.

(4) Department of Biology, Payame Noor University, I.R. of IRAN.

(5) Department of Horticulture, Science and Research Branch, Tehran, Islamic Azad University, Tehran, Iran.

(6) Department of Clinical Biochemistry, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

(7) Young Researchers and Elites Club, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran.

Corresponding Author: Masoud Negahdary, Young Researchers and Elites Club, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran

E-mail: masoudnegahdary@yahoo.com
Table 1: zymogene fermentation of saccharomyces cerevisiae including
AAT Gen along with starting of inspiration at 2 after insemination
with IPTG0.4m M and cycle enginge which is depending on oxygen

                   Time        The time
                   according   after                 Speed on
explanation        to hours    inspiration   OD600   minute

100[micro]g/ml     1           0             0.07    150
  ampicilin        2           1             0.1     150
mM0.4IPTG          3           2             0.309   150
  100[micro]g/ml   4           3             0.62    200
  ampicilin        5           4             0.71    210
                   6           5             0.9     215
                   7           6             1.1     220

                                       Weather
                   Time                pressure
                   according           according
explanation        to hours    Temp.   to vvm      PH

100[micro]g/ml     1           30      1           7
  ampicilin        2           30      1           7
mM0.4IPTG          3           30      1           7
  100[micro]g/ml   4           30      1           7
  ampicilin        5           30      1           7
                   6           30      1           7
                   7           30      1           7

Table 2: zymogene fermentation of saccharomyces cerevisiae
including AAT Gen along with starting of inspiration at 3
after insemination with IPTG0.4m M and cycle enginge which
is depending on oxygen

                 Time        The time
                 according   after                  Speed on
explanation      to hours    inspiration   OD 600   minute

100[micro]g/ml   1           0             0.1      150
  ampicilin      2           1             0.3      150
                 3           2             0.45     179
mM0.4IPTG100     4           3             0.69     185
  [micro]g/ml    5           4             0.80     203
  ampicilin      6           5             1.11     236
                 7           6             1.3      254
                 8           7             1.5      273

                                     Weather
                 Time                pressure
                 according           according
explanation      to hours    Temp.   to vvm       PH

100[micro]g/ml   1           30      1            7
  ampicilin      2           30      1            7
                 3           30      1            7
mM0.4IPTG100     4           30      1            7
  [micro]g/ml    5           30      1            7
  ampicilin      6           30      1            7
                 7           30      1            7
                 8           30      1            7

Table 3: zymogene fermentation of saccharomyces cerevisiae
including AAT Gen along with starting of inspiration at 4
after insemination with IPTG0.4m M and cycle enginge which
is depending on oxygen

                   Time         The time
                   according    after                  Speed on
explanation        to hours     inspiration   OD 600   minute

100[micro]g/ml     1            0             0.1      150
  ampicilin        2            1             0.3      150
                   3            2             0.7      180
                   4            3             0.85     195
mM0.4IPTG          5            4             0.82     220
  100[micro]g/ml   6            5             1.2      260
  ampicilin        7            6             1.56     270
                   8            7             1.61     280
                   9            8             1.8      320

                                        Weather
                   Time                 pressure
                   according            according
explanation        to hours     Temp.   to vvm       PH

100[micro]g/ml     1            30      1            7
  ampicilin        2            30      1            7
                   3            30      1            7
                   4            30      1            7
mM0.4IPTG          5            30      1            7
  100[micro]g/ml   6            30      1            7
  ampicilin        7            30      1            7
                   8            30      1            7
                   9            30      1            7

Table 4: zymogene fermentation of saccharomyces cerevisiae
including AAT Gen along with starting of inspiration at 5
after insemination with IPTG0.4m M and cycle enginge which
is depending on oxygen

                   Time         The time
explanation        according    after                  Speed on
                   to hours     inspiration   OD 600   minute

100[micro]g/ml     1            0             0.2      150
  ampicilin        2            1             0.4      180
                   3            2             0.7      205
                   4            3             0.9      215
                   5            4             1.3      255
mM0.4IPTG          6            5             1.77     290
  100[micro]g/ml   7            6             1.80     310
  ampicilin        8            7             1.90     335
                   9            8             2.2      355
                   10           9             2.3      390

                                        Weather
                   Time                 pressure
explanation        according            according
                   to hours     Temp.   to vvm       PH

100[micro]g/ml     1            30      1            7
  ampicilin        2            30      1            7
                   3            30      1            7
                   4            30      1            7
                   5            30      1            7
mM0.4IPTG          6            30      1            7
  100[micro]g/ml   7            30      1            7
  ampicilin        8            30      1            7
                   9            30      1            7
                   10           30      1            7

Table 5: zymogene fermentation of saccharomyces cerevisiae
including AAT Gen along with starting of inspiration at 6
after insemination with IPTG0.4m M and cycle enginge which
is depending on oxygen

                   Time         The time
                   according    after                  Speed on
explanation        to hours     inspiration   OD 600   minute

100[micro]g/ml     1            0             0.12     150
  ampicilin        2            1             0.4      170
                   3            2             0.65     190
                   4            3             1.107    200
                   5            4             1.6      266
                   6            5             1.8      280
mM0.4IPTG          7            6             1.9      290
  100[micro]g/ml   8            7             1.95     300
  ampicilin        9            8             2.3      325
                   10           9             2.4      300
                   11           10            2.41     390

                                        Weather
                   Time                 pressure
                   according            according
explanation        to hours     Temp.   to vvm       PH

100[micro]g/ml     1            30      1            7
  ampicilin        2            30      1            7
                   3            30      1            7
                   4            30      1            7
                   5            30      1            7
                   6            30      1            7
mM0.4IPTG          7            30      1            7
  100[micro]g/ml   8            30      1            7
  ampicilin        9            30      1            7
                   10           30      1            7
                   11           30      1            7

Table 6: zymogene fermentation of saccharomyces cerevisiae
including AAT Gen along with starting of inspiration at 7
after insemination with IPTG0.4m M and cycle enginge which
is depending on oxygen

                    Time         The time
                    according    after                  Speed on
explanation         to hours     inspiration   OD 600   minute

100[micro]g/ml      1            0             0.12     150
  ampicilin         2            1             0.40     170
                    3            2             0.65     190
                    4            3             1.180    200
                    5            4             1.60     270
                    6            5             1.80     280
                    7            6             1.85     290
mM0.4IPTG           8            7             2.15     300
  100[micro]g/ml    9            8             2.30     325
  ampicilin         10           9             2.34     350
                    11           10            2.5      389
                    12           11            2.85     390

                                         Weather
                    Time                 pressure
                    according            according
explanation         to hours     Temp.   to vvm       PH

100[micro]g/ml      1            30      1            7
  ampicilin         2            30      1            7
                    3            30      1            7
                    4            30      1            7
                    5            30      1            7
                    6            30      1            7
                    7            30      1            7
mM0.4IPTG           8            30      1            7
  100[micro]g/ml    9            30      1            7
  ampicilin         10           30      1            7
                    11           30      1            7
                    12           30      1            7
COPYRIGHT 2013 American-Eurasian Network for Scientific Information
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2013 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Majdi, Sahar; lotfi, Abbas Sahebghadam; siahmazgi, Maryam Ghodrati; Chelongar, Reyhaneh; Behrooznam,
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:7IRAN
Date:Nov 1, 2013
Words:3787
Previous Article:A comparison of the effect of aquatic and land exercise on the general health of people with a disability.
Next Article:To determine and examine the prospect and feasibility study of anti-cancer drugs for local production in Bangladesh.
Topics:

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters