Cloning, Expression and Activity Analysis of a Bacterial Serine Protease
选修 8 unit 2 cloning
The new plants that grow from the runners （蔓藤植物）are exact copies of the original plant.
Twins
They are born naturally and identical in sex and appearance.
Cloning can help save
endangered animals Cloning can provide a copy of a child for a couple whose child has died
Quiz
In pairs, look at these pictures and discuss which ones are natural clones and which ones are man-made. Think about how they differ.
A strawberry plant
The writer’s attitude towards cloning
A. Objective (be fair) B. Negative (be against it) C. Positive (be in favour of it)
the author does not state any personal Because______________________________ opinion about cloning.
3. What problems may arise when humans are cloned? • People may want to clone themselves so they can live forever. • People may want to clone dead children. • People may want to clone their favourite pets.
cloning英语作文
cloning英语作文Cloning is a controversial topic that has sparked heated debates among scientists, ethicists, and the general public. The idea of creating genetically identical copies of living organisms has raised ethical, moral, andpractical concerns.Some people argue that cloning can be used to replicate valuable genetic traits in plants and animals, leading to increased food production and disease resistance. They believe that cloning technology has the potential to revolutionize agriculture and help address the global food crisis.On the other hand, opponents of cloning raise concerns about the welfare of cloned animals, questioning whether they would suffer from health issues and abnormalities. They also worry about the potential for exploitation and abuse of cloning technology, such as creating designer pets or even human clones.From a scientific perspective, cloning has thepotential to advance our understanding of genetics and developmental biology. By studying the process of cloning, researchers can gain insights into how cells differentiate and specialize, which could lead to new medical treatments and therapies.However, the prospect of human cloning raises profound ethical and moral questions. Many people find the idea of creating genetically identical copies of humans deeply unsettling, fearing the potential for identity confusion, psychological harm, and exploitation.In conclusion, cloning is a complex and multifaceted issue that elicits strong emotions and diverse perspectives. While it holds promise for scientific advancement and practical applications, it also raises profound ethical, moral, and practical concerns that must be carefully considered and addressed.。
《基因工程》习题及参考答案
《基因工程》习题及参考答案一、习题：1. What are biotechnology and genetic engineering?2. What is a gene?3. What are genetically engineered medicines?4. What do genome research and human genetics deal with?5. What potentials are held out by genetic diagnosis?6. What options are given by gene therapy?7. What is an embryo - and what is a fetus?8 What is a genetic fingerprint?9 What does the term "therapeutic cloning" mean?10 What are stem cells?11 What is a transgenic organism?12 What does xenotransplantation mean?13 How will genetic engineering be used in agriculture?14 How are genetically modified organisms assessed?15 What does the German Embryo Protection Act regulate?16. What is a genome?17. Is there a risk of bioterrorism?18. How does genetic engineering affect the environment?19. Are genetically engineered crops good for farmers?20.What is the difference between restriction digestion and restriction mapping?21.Can you combine two different restriction enzymes in the same reaction tubes todigest the DNA molecules?22.Why should we need to generate restriction mapping data?23.How many restriction enzymes available now on the market?24.Why do you consider mutagenesis in vitro as one of the most critical techniquesfor us to understand in genetic engineering class?25.How do we choose the methods for DNA modification?26.How do we choose a gene expression system?27.How can we express eukaryotic gene in E.coli?28.What should we consider before we start the recombinant protein expressionexperiment?29.What is the advantage of yeast expression system?30.What is the advantage of insect expression system?31.Why there are so many different types of vectors available for cloning?32.What is the difference between cloning vector and expression vector?33.What is a genetic fingerprint?34. 基因具体分成多少种类？35. 什么叫印记基因？36．什么叫遗传漂变？37．人类基因组图谱和初步分析结果是在哪一年公布的？38．人类基因组共有多少基因?39. 克隆羊成功的技术关键是什么？40. 有人计划将两个不同物种的动植物体细胞进行融合，然后将融合体的核移植到其中一种生物的未受精卵细胞中，进行体细胞克隆。
Cloning, expression and purification of the general stress protein Yhbo from Escherichia co
CLONING, EXPRESSION AND PURIFICATION OF THE GENERAL STRESS PROTEIN YhbO FROM ESCHERICHIA COLIJad Abdallah1, Renee Kern1, Abderrahim Malki, Viola Eckey, Gilbert Richarme*Stress molecules, Institut Jacques Monod, Universite Paris 7, 2 place Jussieu, 75005 Paris, France*Corresponding author1The two first authors contributed equally to the work, and their name was arbitrarily choosen.Tel 33 1 44 27 50 98Fax 33 1 44 27 57 16Email: richarme@ccr.jussieu.frAbbreviations: Hsp: heat shock protein˚; amc˚: 7-amino-4-methylcoumarin˚; DTT˚: dithiothreitol.ABSTRACTWe cloned, expressed and purified the Escherichia coli yhbO gene product, which is homolog to the Bacillus subtilis general stress protein 18 (the yfkM gene product), the Pyrococcus furiosus intracellular protease PfpI, and the human Parkinson disease protein DJ-1. The gene coding for YhbO was generated by amplifying the yhbO gene from E. coli by polymerase chain reaction. It was inserted in the expression plasmid pET-21a, under the transcriptional control of the bacteriophage T7 promoter and lac operator. A BL21(DE3) E. coli strain transformed with the YhbO-expression vector pET-21a-yhbO, accumulates large amounts of a soluble protein of 20 kDa in SDS-PAGE that matches the expected YhbO molecular weight. YhbO was purified to homogeneity by HPLC DEAE ion exchange chromatography and hydroxylapatite chromatography and its identity was confirmed by N-terminal sequencing and mass spectrometry analysis. The native protein exists in monomeric, trimeric and hexameric forms.INTRODUCTIONEscherichia coli YhbO is a member of the DJ-1/ThiJ/Pfp1 superfamily, which includes proteins with diverse functions, chaperones (E. coli Hsp31) (1-4), proteases (Pfp1 from Pyrococcus furiosus (5) and PHP1 from Pyrococcus horikoshii (6)), catalases, the Parkinson disease protein DJ-1 (7), and the ThiJ kinase involved in thiamine biosynthesis (8). The closest YhbO homologs are ThiJ in E. coli, YfkM and YraA in B. subtilis (involved in protection against environnemental stresses (9, 10)), the Pyrococcus furiosus protease 1 (5), and the Parkinson disease protein DJ-1 (for which different functions have been proposed, i.e. chaperone, peptidase, oxidative stress sensor) (7, 11, 13). The crystal structure of several members of the ThiJ superfamily has been solved. All members of the superfamily contain a similar domain with a nucleophilic elbow displaying an important cysteine, which is part, in Php1 and Hsp31, of a Cys, His, Glu/Asp catalytic triad responsible for their peptidase activity (3, 6). YhbO also possesses such a putative catalytic triade whereas DJ-1 contains instead a Cys, Glu diad (7).Several members of this superfamily have been biochemically characterized. Hsp31 was characterized as a chaperone (3, 4) and a peptidase (14), and Pfp1 as a protease / peptidase active against gelatin and the fluorescent substrate AAF-amc (5). The biochemical characterization of DJ-1 led to contradictory results concerning putative chaperone, peptidase or redox activities (11, 13). The exact function of ThiJ in thiamine biosynthesis is not yet known.Most of the members of the ThiJ superfamily function in cellular protection against environnemental stresses. The chaperone/peptidase Hsp31 is involved in thermal stress protection (2), YfkM and YraA in Bacillus subtilis protection against multiple stresses (9, 10), and DJ-1 in cellular protection against oxidative stress (13). E. coli YhbO is several fold overexpressed in stationary phase, during hyperosmotic stress or acid stress (15), and an yhbO-deficient strain is more sensitive than the parental strain to thermal, oxidative, hyperosmotic, pH, and UV stresses (manuscript in preparation), suggesting that YhbO is a general stress protein. In the present work, we report the cloning, expression and purification of Escherichia coli YhbO.MATERIALS AND METHODSBacterial strains, plasmids, and growth conditons.The E. coli BL21 (DE3) strain (Novagen, USA) was used for the transformation of the synthetized DNA and the expression of YhbO. The gene coding for YhbO was generated by amplifying the yhbO gene from E. coli DNA by PCR using the forward primers (5GGTGGTTGCTCTTCACATATGAGTAAGAAAATTGCC-3) containing a Nde1site and the reverse primer (5GGTGGTCTGGGATCCTCATCAGGCAACAGACAGGCG-3) containing a BamH1site. The resulting product was digested with Nde1 and BamH1, ligated to the pET-21a (Novagen, USA) Nde1 and BamH1 backbone fragment, and transformed into strain BL21 (DE3).Bacterial extracts preparation.The YhbO overproducing strain (BL21 (DE3) pET-21a-yhbO was grown at 37¡C in 1 liter of Luria-Bertani medium (16) supplemented with ampicillin (50 g/ml) to an OD600= 0.5. YhbO overexpression was induced with 1 mM IPTG and growth was continued until OD600= 3. Cells were harvested by centrifugation at 4¡C. The cell pellet was resuspended in 10 ml of 30 mM Tris pH 8.0, 20 mM NaCl, 1 mM DTT, 0.5 mM EDTA, and bacteria were disrupted at 0¡C using a Branson sonicator (microtip, level 5, 5 x 30 seconds). This extract was centrifuged for 1 hour at 200,000 x g, and the supernatant was immediately loaded onto a HPLC DEAE column.HPLC DEAE ion exchange chromatography.YhbO was loaded on a TSK-DEAE-5PW (Tosohaas, Germany) equilibrated in 30 mM Tris pH 8.0, 20 mM NaCl, 1 mM DTT, 0.5 mM EDTA at 20¡C, and it was eluted (around 200 mM NaCl) with a linear gradient of 0.03 — 1 M NaCl in the same buffer. Fractions were analyzed by SDS-polyacrylamide gel electrophoresis and quantified by the Bradford assay.Hydroxyapatite chromatography.Purified fractions from the DEAE column were loaded on a 15 ml hydroxyapatite column (Bio-Gel HTP from BioRad), equilibrated in 20 mM Tris pH 7.5, 50 mM KCl, 1 mM DTT and eluted with a linear gradient of 0 - 100 mM sodium phosphate pH 7.5 in the same buffer. YhbO was dialyzed against 20 mM Tris pH 7.4, 50 mM KCL, 0.5 mM dithiothreitol and stored at —20¡C in buffer supplemented with 30% glycerol.Native molecular weight determination.The molecular weight of native YhbO was determined by filtration of the protein on a TSK-G4000-SW HPLC gel permeation column (Tosohaas, Germany). The column was equilibrated in 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM DTT at 20¡C, loaded with 20 l of purified YhbO (2 mg/ml) and eluted at a flow rate of 0.5 ml/min. YhbO was detected by its absorbance at 280 nm. Alcohol dehydrogenase (molecular weight, 150,000), bovine serum albumin (molecular weight, 66,000), carbonic anhydrase (molecular weight, 29,000) and E. coli thioredoxin 1 (molecular weight, 12,000) were used as molecular weight standards, and were obtained from Sigma.Sodium dodecylsulfate-polyacrylamide gel electrophoresis.We perfored electrophoresis according to Laemmli, using 8-16% polyacrylamide gradient gels (Bio-Rad) with Coomassie blue staining. Broad range molecular weight markers were form Bio-Rad.Protein sequencing.N-terminal sequencing was performed by automated Edman degradation at the Laboratoire de Microsequen age des Prot ines, Pasteur Institute, Paris, with an Applied Biosystems 470 gas phase sequencer and an Applied Biosystems 1000S detector.Mass spectrometric identification of YhbO.Excised YhbO gel band was in-gel digested with mass spectrometry grade trypsin (Roche). Mass spectra were recorded in positive ion reflection mode of a matrix assisted laser desorption ionization-time of flight (MALDI-ToF) Voyager DE PRO (Applied Biosystems). Petide mass obtained were searched against E. coli database using the Mascot engine available online ().Protease, peptidase and chaperone assays.Endopeptidase and aminopeptidase activities were assayed by monitoring the production of 7-amino-4-methylcoumarin (amc) from the fluoregenic aminoacids peptides Suc-LLVY-amc, Boc-LAR-amc, Ac-YVAD-amc, AAF-amc, ALK-amc, L-Ala-amc, L-Arg-amc, L-Asp-amc, L-Asn-amc, L-Gly-amc, L-Leu-amc, L-Lys-amc, L-Met-amc, L-Phe-amc, L-Pro-amc, L-Thr-amc, L-Tyr-amc, L-Val-amc, L-Ser-amc, L-Cys-amc, L-Glu-amc, L-His-amc as described in (14, 17). Carboxypeptidase activity was assayed by measuring the hydrolysis of Hippuryl-Phe or Hippuryl-Arg as described in (14): Gelatin-PAGE and casein-PAGE in-gel proteolysis were performed as described in (1, 5). Renaturation of urea-unfolded citrate synthase and protection of citrate synthase against aggregation at 43¡C were performed as described in (18).Reagents.The Klenow DNA polymerase was from Roche (Mannheim Germany), restriction enzymes were from Invitrogen, and the plasmid extraction kits was from Quiagen. Fluorogenic peptide substrates were from Bachem or Sigma, and all other chemicals were from Sigma and were reagent grade.RESULTS AND DISCUSSIONConstruction of the YhbO-expression vector pET21a-yhbO.The yhbO gene from E. coli was amplified by PCR, with a Nde1 and a BamH1 site at the 5 and 3 end, respectively. The amplified product was about 550 bp (not shown), which is in accordance with the theoretical length of the yhbO gene (519 bp). It was cut wih Nde1 and BamH1, ligated to the pET21a N d e1and BamH1backbone fragment and transformed into strain BL21 (DE3) by electroporation. The constructed plasmid was verified by DNA sequencing (not shown).Expression of YhbOThe BL21 (DE3) strain, transformed with the recombinant expression vector pET-21a-yhbO, and induced with 1 mM IPTG accumulates high amounts of a soluble protein migrating in SDS-PAGE with an apparent molecular weight of 20 kDa (Figure 1, lane 2). The overexpressed protein is not detected in an uninduced extract (Figure 1, lane 1), and represents 28% in mass of the total proteins of the induced extract. This 20 kDa molecular weight matches the expected YhbO molecular weight of 19 kDa. Thus, YhbO migrates exclusively as a monomer in SDS polyacrylamide gels, in contrast with its Pyrococcus furiosus PfpI and Pyrococcus Horikoshii PhpI homolog which both display multmeric forms in SDS polyacrylamide gels (5, 6). YhbO was found in the soluble bacterial extract (200,000 x g supernatant), but not in the 200.000 x g pellet (not shown), suggesting that it neither forms inclusion bodies during overexpression nor does it colocalize with membrane fractions. The cytoplasmic localization of YhbO is in accordance its primary structure which does not contain any signal or membrane spanning sequence.YhbO purificationYhbO was purified to homogeneity by two chromatographic steps on a HPLC DEAE ion exchange column and on a hydroxyapatite column (Figure 1, lane 3 and 4, respectively). Although YhbO appears pure after the first column, we performed a second chromatographic step in order to avoid a single-step purification procedure based on ion exchange chromatography. Since the protein was massively overexpressed under the T7 promoter (around 30% of total protein), overall purification was 3.5 fold, and yield was 77% (Table 1). The identity of YhbO was confirmed by N-terminal sequencing and mass spectrometry. The N-terminal sequence found was SKKIAVLI, which is identical to the YhbO N-terminal sequence without its N-terminal methionine (processing of the N-terminal methionine frequently occurs in bacteria). This sequence is not found in any of the other E. coli proteins. Mass spectrometry analysis identified unambiguously the purified protein as YhbO (Figure 2A and 2B).Quarternary structureThe purified protein was analyzed by size exclusion chromatograhy on a SW G-4000 HPLC column equilibrated in 30 mM Tris pH 7.5, 100 mM NaCl, 1 mM DTT at 20¡C, as described in Materials and Methods. YhbO (20 l, 2 mg/ml) was loaded onto the column at a flow rate of 0.5 ml/min. It elutes under several peaks at 7.77 (expected molecular weight, 118 kDa), 8.88 (expected molecular weight, 58 kDa) and 10.23 min (expected molecular weight, 20 kDa) (Figure 3A). These peaks likely represent hexameric, trimeric and monomeric forms of YhbO, respectively (Figure 3B). Multimeric forms of PfpI-like proteases are commonly found (5, 6), and the crystal structure of PhpI shows a hexameric barrel-like oligomeric structure with the active sites sequestered inside the barrel (6).Absence of proteolytic, peptidolytic or chaperone activity in vitroWe could not detect any proteolytic activity of YhbO, using an SDS/PAGE in-gel assay (used for the detection of the proteolytic activity of PfpI (5)) with gelatin or casein as substrate, or using the highly sensitive BODIPY-casein fluorescent test (11) (not shown). YhbO did not hydrolyze any of the 18 aminoacyl-amc substrates tested, in contrast with PepN (17) or Hsp31 (14). It did not hydrolyze either AlaAlaPhe-amc (the best substrate of Pyrococcus furiosus protease I (5)), the endopeptidase substrates acetyl-Ala-amc and Suc-AlaAlaAla-amc, and the carboxypeptidase substrates Hippuryl-Phe or Hippuryl-Arg (not shown).Since YhbO presents some analogy with the Hsp31 chaperone/peptidase, we checked whether it displays chaperone properties. In contrast with Hsp31 (1), 20 M YhbO was unable to stimulate the renaturation of 0.2 M citrate synthase after denaturation in 8M urea (not shown), or to prevent citrate synthase aggregation upon thermal shock at 43¡C (not shown).ImplicationsWe cloned, overexpressed and purified to homogeneity E. coli YhbO. The overexpressed protein is found in the cytoplasm in a highly soluble form. We identified the purified protein by N-terminal sequencing and MALDI-ToF mass spectrometry. The expression and purification procedures in this study have provided a simple and efficient method to obtain pure E. coli YhbO in large quantities. The YhbO protein obtained will be used for further studies of its structure and function. YhbO exist as a monomer, trimer and hexamer like several archeae proteases (Pyrococcus furiosus proteaseI and Pyrococcus horikoshii proteaseI)However, we could not yet detect any proteolytic activity of YhbO using several protein or peptidic synthetic substrates, nor could we detect any chaperone activity, in contrast with Hsp31 which was recently characterized by us and others as a chaperone (1, 2) and a peptidase (14). Several members of the ThiJ domain superfamily still lack a precise biochemical characterization, like the Parkinson disease protein DJ-1, for which contradictory results have beenreported concerning possible chaperone, peptidase and redox activities (7, 8, 11, 13). Thus, the physiological and biochemical characterization of the ThiJ superfamily stress proteins will still require intensive investigations.ACKNOWLEDGEMENTS.The authors wish to thank Dr. Jacques d’Alayer (Institut Pasteur, Paris) for N-terminal sequencing, Dr. Jean-Jacques Montagne for MALDI-ToF mass spectrometry experiments and A. Kropfinger for correction of the English language. This work was supported by grant CR 521090 from the DGA to G.RREFERENCES1 A. Malki, R. Kern, J. Abdallah, G. Richarme. Characterization of the Escherichia coli YedU protein as a molecular chaperone. Biochem. Biophys. Res. Commun. 301 (2003) 430-436.2 M.S. Sastry,.K. Korotkov, Y. Brodsky, F. Baneyx. Hsp31, the Escherichia coli yedU gene product, is a molecular chaperone whose activity is inhibited by ATP at high temperatures. J. Biol. Chem. 277 (2002) 46026-46034.3 P.M. Quigley, K. Korotkov, F. Baneyx, W. G. Hol. The 1.6-A crystal structure of the class of chaperones represented by Escherichia coli Hsp31 reveals a putative catalytic triad. Proc. Natl. Acad. Sci. U S A. 100 (2003) 3137-3142.4 Y. Zhao, D. Liu, W.D. Kaluarachchi, H.D. Bellamy, M.A. White, R.O. Fox. The crystal structure of Escherichia coli heat shock protein YedU reveals three potential catalytic active sites. Protein Sci.12 (2003) 2303-2311.5 I.I. Blumentals, A.S. Robinson, R.M. Kelly. Characterization of sodium dodecyl sulfate-resistant proteolytic activity in the hyperthermophilic archaebacterium Pyrococcus furiosus. Appl. Environ. Microbiol. 56 (1990) 1992-1998.6 X. Du, I.G. Choi, R. Kim, W. Wang, J. Jancarik, H. Yokota, S.H. Kim. Crystal structure of an intracellular protease from Pyrococcus horikoshii at 2-A resolution. Proc. Natl. Acad. Sci. U S A. 97 (2000) 14079-14084.7 S.J. Lee, S.J. Kim, I.K. Kim, J. Ko, C.S. Jeong, G.H. Kim, C. Park, S.O. Kang, P.G. Suh, H.S. Lee, S.S. Cha. Crystal structures of human DJ-1 and Escherichia coli Hsp31, which share an evolutionarily conserved domain. J. Biol. Chem. 278 (2003) 44552-44559.8 S. Bandyopadhyay, M.R. Cookson. Evolutionary and functional relationships within the DJ1 superfamily. BMC Evol. Biol. 4 (2004) 6-12.9 P.D. Thackray, A. Moir, SigM, an extracytoplasmic function sigma factor of Bacillus subtilis, is activated in response to cell wall antibiotics, ethanol, heat, acid, and superoxide stress. J. Bacteriol. (2003) 185:3491-3498.10 A. Petersohn, H. Antelmann, U. Gerth, M. Hecker, Identification and transcriptional analysis of new members of the sigmaB regulon in Bacillus subtilis. Microbiology 145 (1999) 869-80.11 J.A. Olzmann, K. Brown, K.D. Wilkinson, H.D. Rees, Q. Huai, H. Ke, A.I. Levey, L. Li, L.S. Chin,Familial Parkinson's disease-associated L166P mutation disrupts DJ-1 protein folding and function. J. Biol. Chem. 279 (2004) 8506-8515.12 S.B. Halio, I.I. Blumentals, S.A. Short, B.M. Merrill, R.M. Kelly, Sequence, expression in Escherichia coli, and analysis of the gene encoding a novel intracellular protease (PfpI) from the hyperthermophilic archaeon Pyrococcus furiosus. J. Bacteriol. 178 (1996) 2605-2612.13 S. Shendelman, A. Jonason, C. Martinat, T. Leete, A. Abeliovich. DJ-1 is a redox-dependent molecular chaperone that inhibits alpha-synuclein aggregate formation. PLoS Biol. 2 (2004) e362.14 A. Malki, T. Caldas, J. Abdallah, R. Kern, S.J. Kim, S.S. Cha, H. Mori, G. Richarme, Peptidase activity of the Esch erichia coli Hsp31 chaperone. J. Biol. Chem. 280 (2005) 14420-14426.15 H. Weber, T. Polen, J. Heuveling, V.F. Wendisch, R. Hengge,Genome-wide analysis of the general stress response network in Escherichia coli: sigmaS-dependent genes, promoters, and sigma factor selectivity. J. Bacteriol. 187 (2005) 1591-1603.16 J.H. Miller, Experiments in Molecular Genetics, p. 439. Cold Spring Harbour Laboratory, Cold Spring Harbour, NY (1972).17 D. Chandu, A. Kumar, D. Nandi, PepN, the major Suc-LLVY-AMC-hydrolyzing enzyme in Escherichia coli, displays functional similarity with downstream processing enzymes in Archaea and eukarya. Implications in cytosolic protein degradation. J. Biol. Chem. 278 (2003) 5548-5556.18 T. Caldas, A. El Yaagoubi, G. Richarme, Chaperone properties of elongation factor EF-Tu. J. Biol. Chem.273 (1998) 11478-11484.11 LEGENDS TO FIGURESFigure 1. Purification of YhbO. Protein samples were separated by sodium dodecylsulfate polyacrylamide gels (8-16% gradient), and stained with Coomassie brillant blue. Lane 1, crudeextract from BL21(DE3),pET-21a-yhbO uninduced; lane 2, crude extract from BL21(DE3), pET-21a-yhbO induced with IPTG; lane 3, YhbO pool (8 µg) from the HPLC DEAE column, lane 3,YhbO pool (30 µg) from the hydroxyapatite column. The positions of the molecular weight markers(kDa) are indicated on the left.Figure 2. MALDI-ToF mass spectrum of the tryptic digest of E. coli YhbO. A) MALDI-ToF spectrum. B) Matched peptides of YhbO.Figure 3. Native molecular weight determination. Purified YhbO (20 l, 2 mg/ml) was loadedonto a TSK-G4000-SW HPLC gel permeation column as described under Experimental Procedures , and eluted at a rate of 0.5 ml/min. A) Chromatogram of the eluted fractions. Peaksrepresent the absorbance of YhbO at 280 nm. B) Elution time of the molecular weight markers as afunction of the logarithm of their molecular weight: Alcohol dehydrogenase (150,000 Da), bovineserum albumin (66,000 Da), carbonic anhydrase (29,000 Da) and E. coli thioredoxin 1 (12,000 Da).The elution volume of the three YhbO peaks displayed in Figure 3A (at 7.77 min, 8.88 min and10.23 min) are indicated by crosses marqued respectively H, T and M, corresponding to putative hexameric (around 12O kDa), trimeric (around 60 kDa) and monomeric (around 20 kDa) forms ofYhbO.MSKK IAVLITDEFEDSEFTSPADEFR KAGHEVITIEKQAGKTVKGKKGEASVTIDK S I D E60 VTPAEFDALLLPGGHSPDYLR GDNRFVTFTRDFVNSGKPVFAICHGPQLLISADVIRGRK120 LTAVKPIIIDVK NAGAEFYDQEVVVDKDQLVTSRTPDDLPAFNR EALRLLGA172 Figure2B.Matched peptide fragments i n YhbO.The matched portions a r e s h o w n in bold a n d underlined.Purification step Total protein(mg)Total YhbO(mg)Yield (%)Overall purification(fold)200.000 x gsupernatant22062DEAE pool545284 3.4Hydroxyapatitepool484877 3.5Table 1 : Purification of YhbO from Escherichia coli。
Cloning and expression of a full-length glutamate decarboxylase gene fromLactobacillus brevis BH2
Cloning and Expression of a Full-LengthGlutamate Decarboxylase Gene fromLactobacillus brevis BH2=pÉJeÉÉ=háãN I=_çJeóÉ=pÜáåN I=vÉçåJeÉÉ=háãN I=pççJt~å=k~ãNIO I=~åÇ=pìåÖJgçåÖ=gÉçåNIO G=1 Department of Biotechnology and Bioengineering, Dong-Eui University, Busan 614-714, Korea2 Department of Biomaterial Control (Brain Korea 21 Program), Dong-Eui University, Busan 614-714, Korea^Äëíê~Åí=A bacterium (BH2) that was found to produce a large amount of γ-aminobutyric acid (GABA) was isolated from háãÅÜá, a traditional fermented food in Korea. Phylogenetic analysis based on the 16S rDNA sequence and biochemical studies indi-cated that BH2 belonged to the genus i~ÅíçÄ~ÅáääìëÄêÉîáë. Under controlled conditions in MRS broth (Difco) with 5% mono-sodium glutamate, this strain produced GABA at a concentration of 194 mM with a 73% GABA conversion rate after 48 h. A full-length glutamate decarboxylase (Ö~Ç) gene was cloned by the rapid amplification of cDNA ends (RACE) PCR. The open reading frame (ORF) of the Ö~Ç gene was composed of 1,407 nucleotides and encoded a protein (468 amino acids) with a predicted molecular weight of 53.5 kDa. The deduced amino acid sequence of GAD from iKÄêÉîáë showed 97.5 and82.7% identities to the iK=ÄêÉîáë OPK-3 GAD and iK=éä~åí~êìã WCFS1 GAD, respectively. The Ö~Ç gene was expressed inbëÅÜÉêáÅÜá~=Åçäá cells and the expression was confirmed by SDS-PAGE analysis and enzyme activity studies. © KSBBhÉóïçêÇëW=Lactobacillus brevis, γJ~ãáåçÄìíóêáÅ=~ÅáÇ=Ed^_^F, Öìí~ã~íÉ=ÇÉÅ~êÄçñóä~ëÉ=Ed^aF, o^`bJm`o=====fkqolar`qflk=Glutamate decarboxylase (GAD: EC 4.1.1.15) is a pyri-doxal 5′-phosphate (PLP)-dependent enzyme, which cataly-ses the irreversible decarboxylation of L-glutamate to γ-aminobutyric acid (GABA). GABA is a non-proteineaceous amino acid known to be a major inhibitory neurotransmitter in the mammalian brain tissues [1,2]. GABA has several physiological functions such as neurotransmission, hypoten-sive activity, diuretic, as well as tranquilizing effects, par-ticularly with regard to insomnia, depression and autonomic disorders observed during the menopausal or presenium pe-riods [3,4]. A recent study indicated that GABA strongly induces insulin secretion from the pancreas [5] and ef-fectively prevents diabetic conditions [6]. Therefore, GABA has the potential to be utilized extensively in the food and pharmaceutical fields.GAD and GABA are widely distributed among bacteria, higher plants and animals [7]. Unlike in animals or bacteria, GABA production in plants is induced by binding of calmodulin to Ca2+, which activates GAD [8]. In bacteria,G`çêêÉëéçåÇáåÖ=~ìíÜçêTel: +82-51-890-2278 Fax: +82-51-890-2632e-mail: jeon.sj@deu.ac.kr the roles of GAD and GABA are not clearly elucidated, but some reports indicate that GABA is functionally involved in the spore budding process of the Bacillus megaterium [9]. It has also been reported that GABA is required for maintain-ing resistance to an acidic pH in Lactococcus lactis and Es-cherichia coli (E. coli) [10,11].Recently, much interest has been generated around the utilization and mass production of GABA as a bioactive component for food. GABA green tea, gabaron tea, and red mold rice have been reported to exert antihypertensive ef-fects in human subjects [12-14]. A mixed culture of Strep-tococcus thermophilus and Lactobacillus delbreuckii, iso-lated from commercially available yoghurt, produced a large amount of GABA [15]. Currently, various lactic acid bacteria (LAB), Lb. plantarum[16], Lb. brevis[17], Lb. paracasei[18],Lb. sakei[19], have also been found to produce a large amount of GABA. However, the functions and properties of GABA and GAD in LAB are still un-known.In this study, we isolated a new GABA-producing micro-organism from Kimchi, a traditional fermented food in Korea. This bacterial strain, BH2, was identified as Lactobacillus brevis. In addition, we cloned the gene encoding a GAD from the isolated strain and determined an activity of the recombinant protein in E. coli.TMU=j^qbof^ip=^ka=jbqelap==_~ÅíÉêá~ä=píê~áåë=~åÇ=mä~ëãáÇë=The LAB producing GABA was isolated from Kimchi and formed a clear zone on MRS (Difco) agar plates con-taining 2% (w/v) CaCO3at 30o C for 24 h. The isolated strains were incubated in MRS [20] medium containing 5% monosodium glutamic acid (MSG) at 30o C for 48 h. The cells were centrifuged, and the supernatant was analyzed for formation of GABA by using thin-layer chromatogra-phy (TLC) [21]. Microorganisms that produced high levels of GABA were selected and identified by studying their biochemical properties using an API kit 50CHL (Bio-Mériux Co., France), and by 16S rDNA sequence determi-nation [22].E. coli DH5α and BL21 (DE3) codon plus (Novagen, Inc., San Diego, CA, USA) were used as the cloning and expres-sion host cells, respectively. Plasmid pGEM-T vector (Pro-mega, Madison, WI, USA) was used for DNA cloning and sequencing, and pET-32a (Novagen) was used for the ex-pression of GAD protein.`äçåáåÖ=~åÇ=ak^=pÉèìÉåÅáåÖ==DNA fragments containing the gad gene were obtained by the method of the 5′rapid amplification of cDNA ends (RACE) and 3′RACE PCR. Initially the core fragment of the gad gene was amplified by PCR using the chromosomal DNA as template and two primers, GAD1: 5′-TCGAGA-AGCCGATCGCTTAGTTCG-3′(forward) and GAD2: 5′-TATTGTCCGGTATAAGTGATGCCC-3′(reverse), which were designed from highly conserved regions of GAD [16,23]. Amplification by PCR was carried out at 94o C for 30 sec, 55o C for 30 sec, 72o C for 1 min for 30 cycles using Taq DNA polymerase (Takara, Japan). The PCR product was purified using the GENEALL PCR Purification Kit (General biosystem, Seoul, Korea), ligated into pGEM-T vector by T4 DNA ligase, and then transformed into E. coli DH5α. The cloned DNA was sequenced using an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer).Both 5′and 3′RACE PCR were performed with gene-specific primers designed with partial sequences obtained by PCR. The primer sequences were as follows: 5′-RACE, 5′-CGGCTGAATCTCGCCACGTTCTGT-3′and nested primer, 5′-TCACAGAAGTATGCCAAGCGA-3′; 3′-RACE, 5′-GCTGGCAGGTTCCCACCTATCCC-3′ and nested pr-imer, 5′-TCACAGAAGTATGCCAAGCGA-3′. The first products were amplified with RACE nested primer and the purified products were used as templates for a second PCR reaction with the nested primer using the DNA Walking Speed Up kit (Seegene, Korea) as previously described [24].bñéêÉëëáçå=çÑ=íÜÉ=Ö~Ç=dÉåÉ=To examine gad expression, a DNA fragment containing gad was amplified by PCR using two primers (primer 1, 5′-GCT ATG TTG TAT GGA AAA CA-3′; primer 2, 5′-CGG GAT CCT TAG TGC GTG AAC CCG TAT TT-3′fication by PCR was carried out at 94o C for 30 sec, 58o C for 30 sec, 72o C for 1 min for 30 cycles using Taq DNA poly-merase (Takara, Japan). The expression vector pET-3d (No-vagen, Madison, WI, USA) was digested with Nco I, treated with T4DNA polymerase to fill in the cohesive ends and again digested with Bam HI. The amplified PCR product was digested with Bam HI (the Bam HI site in primer 2 is under-lined) and inserted into the pET-3d. The resulting plasmid was designated as pET-GAD.E. coli codon plus cells harboring the plasmid pET-GAD were grown in 2 × YT medium (1% yeast extract, 1.6% tryptone, 0.5% NaCl) containing ampicillin (100 μg/mL) to an OD600of approximately 0.6 and then gene expression was induced with 1 mM IPTG for 16 h at 25o C. The cells were centrifuged and the pellet was washed with resuspen-sion buffer (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 1 mM DTT, 5 mM MgCl2, 10% glycerol, 1 mM PLP). The cells were then disrupted by sonication, and the supernatant frac-tion was recovered by centrifugation at 14,000 × g for 30 min at 4o C. The supernatant was used for the analysis of GAD activity and SDS-PAGE (Sodium dodecyl sulphate −polyacrylamide gel electrophoresis) [25]. Protein concen-tration was determined using Bio-Rad protein assay system (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as the standard.qÜáå=i~óÉê=`Üêçã~íçÖê~éÜó=Eqi`F=^å~äóëáë= GABA was qualitatively analyzed using TLC with a cellu-lose F aluminum plate (Merck Co., Germany). A culture fluid was centrifuged at 1,500 × g for 15 min, and 5 μL of supernatant was then spotted onto TLC plates with n-butanol-acetic acid-water (4:1:1, by volume) [21]. The prod-ucts were detected by spraying the plates with 0.2% ninhy-drin solution and treating it at 110o C for 10 min.eáÖÜJéÉêÑçêã~åÅÉ=iáèìáÇ=`Üêçã~íçÖê~éÜó=Eemi`F= ^å~äóëáë=The enzyme solution was incubated with 5% MSG in 50 mM phosphate buffer (pH 7.0), at 30o C for 10 min, and the reaction was stopped by boiling for 5 min. The reaction mix-ture was derivatized with PITC (phenylisothiocyanate) [26] and then filtered through a 0.45 μm Millipore filters, and analyzed by HPLC (Waters alliance 2690). Chromatography was performed using a Nova-Pak C18HPLC column (150 mm × 3.9 mm), on a Waters alliance 2690 system and a Wa-ters model 2487 detector (controlled at 254 nm filter) [27]. Separation of the derivatized samples was accomplished with a binary non-linear gradient using eluant A (0.14 M sodium acetate, 10 mM EDTA (ethylenediaminetetraacetic acid) and 0.5 mL TEA (triethylamine) per liter, titrated to pH 6.4 with acetic acid mixed with 6% acetonitrile) and eluant B (60% acetonitrile containing 10 mM EDTA). The column temperature was set at 46o C. A Pierce standard amino acid mixture (hydrolysis standard) was used to calibrate the analyses. The authentic GABA and glutamate were used as controls.Biotechnol. Bioprocess Eng. TMV=cáÖK=NK TLC chromatogram of GABA production by the isolated lactic acid bacteria. Lanes 1, GABA standard; 2, MSG inMRS medium; 3, strain SH2; 4, strain SH7; 5, strain SH9;6, strain SH12; 7, strain SH15; 8, strain BH2; 9, strainBH5.obpriqp=^ka=afp`rppflk=fëçä~íáçå=~åÇ=fÇÉåíáÑáÅ~íáçå=çÑ=d^_^JéêçÇìÅáåÖ==jáÅêççêÖ~åáëã=The microorganism producing GABA was isolated from Kimchi. During the screening stage, all microorganisms pro-ducing GABA from MSG were selected. Among the sc-reened microbes, one strain showed a particularly high GABA production by TLC analysis (Fig. 1). The production of GABA was confirmed by HPLC. The isolated strain was identified as a Gram-positive and motility-negative bacte-rium. It was further identified as L. brevis by examining its biochemical characteristics using an API kit 50CHL (data not shown). The 16S rRNA sequence (1,383 bp) of this strain showed the highest identity of 99% to that of L. brevis (GenBank accession no. DQ523492). Therefore, this strain was designated as L. brevis BH2.dêçïíÜI=éeI=~åÇ=d^_^=mêçÇìÅíáçå=çÑ==íÜÉ=iK=ÄêÉîáë=_eO=The growth properties and pH of L. brevis BH2 cell cul-ture in MRS medium at different times of cultivation were investigated. Fig. 2A shows the cell growth and pH changes in MRS with or without 5% MSG. The growth of L. brevis BH2 in MRS media with and without MSG reached a sta-tionary phase after 32 and 20 h of incubation time, respec-tively. The pH of the culture medium without MSG rapidly decreased from 6.2 to 3.7 until stationary phase, compared to the pH of the culture medium containing MSG, which exhib-ited a gradual decrease from 6.3 to 4.8. It has been reported that a proton motive force occurs from the cytoplasmic pro-ton consumption that accompanies glutamate decarboxyla-tion in L. lactis and E. coli, and then the product GABA is exported from the cell via the antiporter; this is believed to contribute toward maintaining a neutral intracellular pH when the external pH drops [28]. The results obtained from ^==_cáÖK=OK Cell growth, pH changes, and GABA production of iK=ÄêÉîáë BH2 in culture media. iK=ÄêÉîáë BH2 was cultivatedfor 48 h at 30o C. (A) Cell growth and pH changes at dif-ferent times of cultivation. Closed triangles, cell growth inMRS medium with 5% MSG; open triangles, cell growthin MRS medium; closed circles, culture pH in MRS me-dium with 5% MSG; open circles, culture pH in MRS me-dium. (B) GABA production yield at different times of cul-tivation. All values are means ± SE.this study are consistent with the possibility that the mainte-nance of pH homeostasis is mediated by glutamate decar-boxylase, as it is in L. lactis and E. coli. The exchange of extracellular glutamate for the more alkaline GABA was postulated to cause the slight decrease in the extracellular pH of the culture medium with MSG.The amount of GABA produced at different times of cul-tivation was measured using HPLC analysis. As shown in Fig. 2B, 5% MSG (266 mM) was converted into 194 mM GABA (a 73% conversion rate) within a 48-h culture period. Komatsuzaki et al. [18] showed that 302 mM GABA was produced from 500 mM glutamate by L. paracasei NFRI 7415 for 150 h. Choi et al. [29] reported that 534 mM MSG was converted into 223 mM GABA by L. brevis GABA 057 after 48 h. In addition, the GABA producing ability of L. brevis BH2 was remarkably higher than that of L. brevis IFO-12005 [30]. In future studies, it would thus be interest-ing to compare the GABA production of L. brevis to that of more closely related the lactic acid bacteria [31].`äçåáåÖ=~åÇ=pÉèìÉåÅÉ=^å~äóëáë=çÑ=íÜÉ=iK=ÄêÉîáë==_eO=Ö~Ç=dÉåÉ==In order to clone the gad gene from L. brevis BH2, the PCR method was used for cloning experiment as mentioned in materials and methods. PCR for the core fragment was carried out using primers designed from highly conservedTNM=cáÖK=PK PCR amplification of Ö~Ç ORF from iK=ÄêÉîáë BH2. M,DN A molecular weight marker; 1, PCR product of the amplified Ö~Ç gene.regions of GAD [16,23]. After the initial PCR reaction, PCR product (537 bp) corresponding to the core fragment was cloned into pGEM-T vector, and sequenced. To obtain the sequence information of the full-length gad -gene, 5′-RACE and 3′-RACE PCR were performed with gene-specific prim-ers designed with partial sequences and nested primers. The PCR product for the entire open reading frame (ORF) of the gad gene was obtained by the PCR, cloned into pGEM-T vector, and sequenced (Fig. 3). The ORF encoded a protein (GAD) comprising of 468 amino acids with a predicted molecular weight of 53.5 kDa and an estimated isoelectric point (pI) of 5.34.Fig. 4 shows schematic drawings of the alignment of L. brevis BH2 GAD with its homologues. The deduced amino acid sequence of GAD from L. brevis BH2 was shown to be the most homologous to that (97.5% identity) from L. brevis OPK-3 [23] and showed a relatively high homology to GAD (82.7% identity) of L. plantarum WCFS1 (Fig. 4) [16]. Both GAD proteins from L. brevis BH2 and OPK-3 contained highly conserved catalytic domains. In contrast, the N-ter-minal region was not conserved and was found to be variable in length (Fig. 4). These glutamate decarboxylases (GADs) are members of the group II decarboxylase family, which belongs to the pyridoxal 5′-phosphate (PLP)-dependent de-carboxylase super-family [32]. As observed for the group II decarboxylase [33,34], the cloned L. brevis BH2 GAD was shown to possess a conserved lysine residue (Lys279) that was required for the binding of the PLP as well as the active site residues (Thr215 and Asp246) that promote decarboxy-lation (Fig. 4). In addition, the motif {H(I/V)DAASGG}, which is particularly conserved in PLP-dependent decar-boxylase was discovered [35]. The nucleotide sequence of the gad gene from L. brevis BH2 has been submitted to the DDBJ/EMBL/GenBank nucleotide sequence database under the accession no. EU084998.cáÖK=QK Alignment of the deduced amino acid sequence of iK=ÄêÉîáë BH2 GAD with its homologues. Cloned, i~ÅíçJ Ä~Åáääìë=ÄêÉîáë BH2 GAD; LbGAD, i~ÅíçÄ~Åáääìë=ÄêÉîáë=OPK-3;=LpGAD, i~ÅíçÄ~Åáääìë=éä~åí~êìã GAD; LlGAD, i~ÅíçÅçÅÅìë=ä~Åíáë GAD; CpGAD, `äçëíêáÇáìã=éÉêÑêáåÖÉåë GAD; EcGAD, bK=Åçäá GAD. Asterisks indicate amino ac-ids conserved among GAD homologues. The boxes rep-resent the H(I/V)DAASGG motif and the PLP binding site. The deduced amino acid sequence was analyzed using the Clustal W (1.83).Biotechnol. Bioprocess Eng. TNN=cáÖK=RK SDS-PAGE analysis of the expression of the iK=ÄêÉîáëGAD in bK=Åçäá. M, Molecular weight markers; lanes 1 and2 are cell extracts of bK=Åçäá transformed with pET-3d andpET-GAD, respectively, after 16 h induction by IPTG.bñéêÉëëáçå=çÑ=íÜÉ=oÉÅçãÄáå~åí=d^a=L. brevis gad was expressed in the pET-3d, as described in materials and methods. The cells harboring pET-GAD were induced by the addition of 1 mM IPTG (isopropyl-1-thio-β-D-galactopyranoside) to the culture media. The extract from E. coli cells transformed with the expression vector lacking any inserted sequence (negative control) was assayed for GAD activity, this was followed by SDS-PAGE analysis with equivalent amounts of protein being loaded. SDS-PAGE analysis showed that the molecular weight of the GAD proteins analyzed was estimated to be about 49.0 kDa (Fig. 5). This value is inconsistent with the size (53,509 Da) calculated from the amino acid sequence. This discrepancy may explained by the many negatively charged amino acid residues of GAD. To further confirm the presence of L. bre-vis GAD in E. coli, the GAD activity in the cell extracts was analyzed by HPLC. The GAD activity of the extract ob-tained from cells expressing pET-GAD was 104.5 nM GABA/min/mg protein, which was greater than 10 times that of the negative control cells (9.6 nM GABA/min/mg protein). These results indicated that the L. brevis BH2 gad gene had indeed encoded GAD.`lk`irpflk=Kimchi is a traditional fermented food in Korea, widely consumed for many decades. Since Kimchi is mainly fer-mented by Lactobacillus sp., its product, GAD has the po-tential to be extensively used in the food and phar-maceutical fields. In this study, we isolated a new GABA-producing microorganism, L. brevis from Kimchi. We have also cloned a full-length gad gene from L. brevis BH2 using the RACE PCR methods, and compared the amino acid se-quence of GAD with the primary structure of GAD proteins from other sources. The protein was successfully expressed in E. coli codon plus cells. The results from this study sug-gested that this strain and recombinant GAD could be used for the industrial production of GABA. Further analysis to understand the biochemical characteristics of this enzyme is currently in progress.^ÅâåçïäÉÇÖÉãÉåíThis work was supported by the Ma-rine and Extreme Genome Research Center Program, Minis-try of Maritime Affairs and Fisheries, Republic of Korea.Received September 19, 2007; accepted November 26, 2007 obcbobk`bp=1. Roberts, E. and S. Frankel (1950) γ-Aminobutyric acidin brain: Its formation from glutamic acid. J. Biol. Chem.187: 55-63.2. Bazemore, A. W., K. A. C. Elliott, and E. Florey (1957)Isolation of factor I. J. Neurochem. 1: 334-339.3. Stanton, H. C. (1963) Mode of action of gamma amino-butyric acid on the cardiovascular system. Arch. Int.Pharmacodyn. 143: 195-204.4. Omori, M., T. Yano, J. Okamoto, T. Tsushida, T. Murai,and M. Higuchi (1987) Effect of anaerobically treated tea (Gabaron tea) on blood pressure of spontaneously hypertensive rats. Nippon Nogeikagaku Kaishi 61: 1449-1451.5. Adeghate, E. and A. S. 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Lee, S. M. Park, M. Y. Lee, G. E. Ji, M.S. Park, and T. R. Heo (2006) Improvement of γ-aminobutyric acid (GABA) production using cell en-trapment of Lactobacillus brevis GABA 057. J. Micro-biol. Biotechnol. 16: 562-568.30.Yokoyama, S., J. I. Hiramatsu, and K. Hayakawa (2002)Production of γ-aminobutyric acid from alcohol distill-ery lees by Lactobacillus brevis IFO-12005. J. Biosci.Bioeng. 93: 95-97.31.Yoo, E. J., H. S. Lim, K. O. Park, and M. R. Choi(2005) Cytotoxic, antioxidative, and ACE inhibiting activities of Dolsan leaf mustard juice (DLMJ) treated with lactic acid bacteria. Biotechnol. Bioprocess Eng.10: 60-66.32.Murzin, A. G. (1996) Structural classification of pro-teins: new superfamilies. Curr. Opin. Struct. Biol. 6: 386-394.33.Burkhard, P., P. Dominici, C. Borri-Voltattorni, J. N.Jansonius, and V. N. Malashkevich (2001) Structural in-sight into Parkinson’s disease treatment from drug-inhibited DOPA decarboxylase. Nat. Struct. Biol.8: 963-967.34.Momany, C., R. Ghosh, and M. 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ABAP面试大全
目录1. 报表知识 (6)1.1基础知识 (6)1.1.1报表事件，有哪些？ (6)1.1.2报表选择画面 (7)1.2ALV报表 (8)1.2.1ALV报表实现的流程 (8)1.2.2显示ALV常用的两个FM (8)1.2.3如何设置ALV中的热键 (8)1.2.4ALV显示中的小计 (8)1.2.5FM ALV 和 OO ALV的比较 (8)1.3WRITE LIST (8)2. 数据库知识 (9)2.1基础知识 (9)2.1.1 ABAP数据字典有哪些对象或元素？ (9)2.1.2 据库提交确认和数据库回滚取消语句 (9)2.1.3 什么是LUW (9)2.1.4简述modify 、insert、update对数据库表做操作时的影响 (9)2.1.5 要描述域、数据元素、表字段之间的关系 (9)2.1.6数据字典有几种缓冲方式，适用范围？ (9)2.2ABAP和数据库 (10)2.2.1 ABAP 数据表的主索引是什么？索引的好处与坏处？与建索引的注意事项！ (10)2.2.2 ABAP透明表有哪几种数据类(data class)？对数据的存储有什么影响？ (10)2.2.3 SAP中有几种表，他们的区别是什么？ (10)2.2.4什么是簇表（cluster table）？举出知道的簇表。
(10)2.2.5找数据库表，有哪些常用的方法。
(10)2.2.6如何建立数据库锁对象，激活锁对象产生的Function Module的名字为什么，在何处查看锁表的情况？ (10)2.2.7更新 FM 分为 V1 和 V2，那么首先会执行哪一种更新类型呢？每种类型又是以哪种模式（异步、同步或本地）执行的呢？ (11)2.2.7使用OPEN SQL注意原则 (11)2.3与表相关 (11)2.3.1 MM模块有哪些常用表格 (11)2.3.2 HR模块知识：HR里面存储HR主数据主要用到了哪些表？ (11)2.3.3 HR模块知识：HR程序在开发中常用的两个逻辑数据库是什么？分别对其进行描述 12 2.3.4 HR模块知识：HR模块里面，如何修改HR的信息类型，具体如何实现 (12)2.3.5财务模块：财务模块开发中常用的表有哪些，简单举例说明： (12)2.3.6PM 常用的TABLE (12)2.3.6inner join 与 left-outer join的区别？ (13)3. 权限相关 (14)3.1什么是权限对象（Authorization Objects）？在 ABAP 程序中使用哪条语句进行授权检查？ (14)3.2与权限对象有关的事务代码有哪些？ (14)4. DIALOG (15)4.1DIALOG 中的几个事件 (15)4.2何在TABLE CONTROL中实现选中一行或多行的效果 (15)4.3DIALOG 开发的常用几个控件是什么？ (15)5. BDC (16)5.1BDC录屏的事务代码 (16)5.2BDC与BAPI之间的区别 (16)5.3BDC录屏的注意事项 (16)5.4谈谈BDC的运行模式和更新模式 (16)6. 增强 (17)6.1什么叫增强？有哪些方式进行增强？ (17)6.1.1 User EXIT (17)6.1.2 Customer exit (17)6.1.3 BADI (17)6.1.4 Enhancement Spot (17)6.2如何建立增强？ (17)6.3与增强相关的事务代码有哪些 (18)6.4如何进行数据库表字段的增强？Append和Include的方式有何区别？ (18)7. SMARTFORMS (19)7.1谈谈SmartForm中，Template和Table表格的区别 (19)7.2SMART FORM如何实现公司LOG打印，其步骤是什么？ (19)7.3smartform 中如何控制段落、单个字符输出格式？ (19)8. RFC和BAPI (20)8.1 RFC (20)8.1.1什么是RFC，有哪些通信模式？ (20)8.1.2 RFC中涉及到常用的事务代码有哪些？ (20)8.1.3根据调用方式的不同，RFC接口提供了什么样的服务？ (20)8.1.4RFC接口的具体功能包括哪些？ (20)8.1.5在通过CALL FUNCTION语句进行远程功能调用的基本模式有哪些 (20)8.1.6怎么创建一个支持远程调用的RFC (21)8.1.7怎么调用一个SAP标准RFC (21)8.1.8怎样建立RFC程序？RFC程序传递的参数都是传递值还是引用？如何建立函数组？ 21 8.1.9怎么来维护这个DESTINATION(远程目标） (21)8.2 BAPI (21)8.2.1什么是BAPI？你使用过哪些BAPI实现什么功能？ (21)8.2.2什么是业务对象类型？它包含哪些主件？ (21)8.2.3如何创建一个BAPI？ (22)8.2.4编写BAPI的注意事项有哪些？ (22)8.2.5谈谈与BAPI相关的事务代码。
Cloning语言点
Cloning语言点重点词汇differ【原句回放】Think about how they differ. 考虑一下它们有怎样的不同。
【点拨】differ vi.有区别，与......相异，不同于常见搭配：differ from 与......不同/有区别（=be different from）differ from/ with 与......意见不一致（=disagree with）differ with sb. on/ about sth. 对某事与某人意见不同differ in 在……方面不同Their house differs from mine in having no garage.他们的房子和我不同，区别在于没有车库。
I differ with him on how to solve the problem. 怎样解决这个问题我和他有分歧。
【拓展】difference n.差异, 差别different adj. 不同的常见搭配：be different from…与……不同make a difference 有差别, 有关系make some (no\ not much\ ...) difference 有（没有\不太大的\......）关系undertake【原句回放】It is difficult task to undertake. 这是一项艰难的任务。
【点拨】undertake (undertook, undertaken) vt. 从事，着手，承担常用搭配：undertake to do sth. 答应/同意做某事undertake that …保证/答应……undertake for ... 为......负责The lawyer undertook the case without a fee. 这位律师免费承办了这个案件。
I undertook to teach the children English. 我答应教孩子们英语。
辣椒COI12基因的克隆、表达分析和植物表达载体的构建
2叫4年27卷4期西南农业学报V01．27No．4Sou山we st C hi n a Jo um al of Agricultural Sciences 1649文章编号：1001—4829(2014)04—1649一07辣椒CD朋．2基因的克隆、表达分析和植物表达载体的构建黄小云，陈再刚，杨俊年，胡廷章4(重庆三峡学院生命科学与工程学院，重庆404000) 摘要：利用RT—PcR技术，通过同源克隆从辣椒中分离到cnc0朋．2基因，采用生物学软件对序列进行生物信息学分析，采用实时定量RT—PcR技术分析基因的表达模式。
结果表明：克隆到的cnc0朋．2基因长度为2041 bp，推测其读码框大小为1812 bp，编码603个氨基酸。
在cac011．2蛋白质N一端有一个F_box结构域，c一端有6个富含亮氨酸结构域。
cac0Ⅱ．2蛋白的氨基酸与已知其他植物c011蛋白序列有53．03％～93．37％的一致性。
聚类分析结果显示：cac011．2与番茄、烟草和葡萄等双子叶植物的亲缘关系较近，而和水稻、玉米、高梁等单子叶植物的亲缘关系较远。
cnc0埘．2在辣椒的不同生长发育时期的组织中都能表达，在花和青熟期果实中表达水平较高，表明饧c0“．2在花和果实的发育过程中起重要作用。
构建植物表达载体pBll21一cac011．2，并导入根癌农杆菌LBA4404中，得到植物转化工程菌，这为下一步用于辣椒等作物的转基因操作、研究cncD盯．2基因在植物中的功能奠定了基础。
关键词：辣椒；c0埘．2；基因克隆；序列分析；基因表达；载体构建中图分类号：S641．3 文献标识码：ACloning and Expression Analysis of CD，J．2 Gene ofChili Pepper and Plant Expression Cassettes ConstructionH U A N G Xiao—yun，CHEN Zai-ga ng，YANG Jun—ni an，H U Ting-z han g+(School of Life Sc ie nc e a nd En gi nee ri ng，C ho ng qi ng Th re e G or ge s Un iv er si ty，C ho ng qi ng404000，C h i n a)A bs t r a ct：I n this p a p er，t h e c D N A se qu en ce s of C。
乌鳢半胱氨酸蛋白酶3基因克隆及表达分析
南方农业学报 Journal of Southern Agriculture 2023，54（11）：3405-3415ISSN 2095-1191； CODEN NNXAABDOI：10.3969/j.issn.2095-1191.2023.11.027乌鳢半胱氨酸蛋白酶3基因克隆及表达分析赵浩航1，2，丘金珠3，周秀莹3，唐怀庆3，赵天珍3，梁富3，宋海霞3，喻大鹏1，2，宋长江3*（1广东海洋大学深圳研究院，广东深圳510000；2广东海洋大学，广东湛江524088；3中山市农产品质量安全检验所，广东中山528400）摘要：【目的】克隆和表达分析乌鳢（Channa argus）的半胱氨酸蛋白酶（Cysteinyl aspartate specific proteinase）3基因（caspase-3），为探究该基因在乌鳢机体中的免疫应答作用及在其他鱼类免疫上的作用提供理论依据。
【方法】根据NCBI公布的乌鳢基因组信息，对乌鳢的caspase-3基因序列进行克隆，对推导的caspase-3氨基酸序列进行生物信息学分析，采用实时荧光定量PCR检测caspase-3基因在健康乌鳢各组织中的表达分布及在不同刺激物［鰤鱼（Seriola quinqueradiata）诺卡氏菌（Nocardia seriolae）、脂多糖（LPS）和聚肌胞苷酸（PolyI：C）］刺激下的时序表达情况。
【结果】caspase-3基因序列全长855 bp，编码284个氨基酸残基，蛋白分子量为31.069 kD，理论等电点（pI）为6.17。
caspase-3蛋白含有1个CASc结构域。
多序列比对结果显示，乌鳢的caspase-3氨基酸序列与秘鲁笛鲷（Lutjanus peru）的caspase-3氨基酸序列相似度最高，达81.7%；系统发育进化树分析结果表明，乌鳢的caspase-3氨基酸序列与秘鲁笛鲷的caspase-3氨基酸序列聚为一支。
关于克隆的辩论的英语作文
关于克隆的辩论的英语作文The Debate on Cloning。
Cloning, the process of creating a geneticallyidentical copy of an organism, has been a topic of heated debate for many years. On one hand, proponents argue that cloning has the potential to revolutionize medical treatment, agriculture, and conservation efforts. On the other hand, opponents raise ethical concerns about the implications of cloning for human identity, individuality, and the natural order of life. In this essay, we will explore both sides of the debate and consider the potential benefits and drawbacks of cloning.Proponents of cloning argue that it has the potential to advance medical treatment in significant ways. For example, cloning could be used to create organs and tissues for transplantation, reducing the need for organ donors and the risk of organ rejection. Additionally, cloning could be used to produce stem cells for regenerative medicine,offering new hope for patients with debilitating diseases and injuries. In the field of agriculture, cloning could be used to produce genetically superior livestock and crops, increasing food production and improving food security. Furthermore, cloning could be used to preserve endangered species and restore biodiversity, contributing to conservation efforts and the protection of ecosystems.However, opponents of cloning raise ethical concerns about the implications of cloning for human identity and individuality. They argue that cloning could lead to a loss of diversity and the commodification of life, as well asthe potential for exploitation and abuse. Furthermore, opponents raise concerns about the potential for unintended consequences and unforeseen risks, such as the emergence of new diseases and the disruption of ecosystems. Additionally, opponents argue that cloning could have a negative impacton society by undermining the natural order of life and the value of human relationships.In conclusion, the debate on cloning is complex and multifaceted, with both potential benefits and drawbacks toconsider. While cloning has the potential to revolutionize medical treatment, agriculture, and conservation efforts, it also raises ethical concerns about the implications for human identity, individuality, and the natural order of life. As the technology and science of cloning continue to advance, it is important for society to engage in thoughtful and informed discussions about the potential benefits and drawbacks of cloning, and to consider the ethical implications of this powerful and controversial technology.。
反对克隆的英语作文
Cloning is a controversial scientific practice that has sparked intense debates worldwide.Here are some points that can be considered when discussing the opposition to cloning:1.Ethical Concerns:Cloning raises numerous ethical issues.It challenges the sanctity of life and the natural process of reproduction.The idea of creating life in a laboratory can be seen as a violation of the natural order.2.Identity and Individuality:Clones would share the same genetic makeup,which could lead to questions about individuality and identity.This could potentially affect their sense of self and their place in society.3.Potential for Abuse:There is a risk that cloning technology could be misused for nefarious purposes,such as creating armies of clones or for the cloning of individuals for organ harvesting.4.Health Risks:The first cloned mammal,Dolly the sheep,had a shorter lifespan and suffered from health issues.This raises concerns about the longterm health of cloned organisms,including humans.5.Social Implications:The introduction of cloned individuals into society could lead to complex social dynamics,including issues of acceptance and the potential for discrimination.6.Legal and Regulatory Issues:There are currently no comprehensive legal frameworks to regulate cloning,which could lead to a lack of oversight and control over the practice.7.Economic and Resource Allocation:The resources required for cloning could be substantial,and there is a debate about whether these resources would be better spent on other areas of scientific research or social welfare.8.Religious and Cultural Objections:Many religious and cultural groups oppose cloning on the grounds that it contravenes their beliefs about the creation of life.9.Unpredictability of Outcomes:The process of cloning is not yet fully understood,and there is a risk of unforeseen consequences,both biological and societal.10.Psychological Impact:The psychological wellbeing of clones could be compromised due to the knowledge of being genetically identical to another individual and the potential pressure to live up to certain expectations.In conclusion,while cloning offers the potential for advancements in medicine and science,the opposition to cloning is grounded in a variety of concerns that span ethical, social,health,and legal domains.It is crucial to weigh these considerations carefully as the scientific community and society at large navigate the implications of cloning technology.。
反对克隆英语作文
Cloning is a controversial topic that has sparked debates around the world.Here are some points that can be used to argue against cloning in an English essay:1.Ethical Concerns:Cloning raises significant ethical issues.It challenges the sanctity of life and the natural process of reproduction.The creation of a cloned individual could be seen as a violation of their right to be unique.2.Potential for Abuse:The technology of cloning could be misused for creating human clones for organ harvesting or other exploitative purposes.This could lead to a devaluation of human life and dignity.3.Health Risks:Cloning techniques are still in their infancy and have not been perfected. Cloned animals have been observed to suffer from various health defects and shorter lifespans.The same risks could apply to humans.4.Identity and Psychological Issues:Cloned individuals might face identity crises and psychological issues due to their genetic similarity with another individual.This could affect their selfesteem and social interactions.5.Impact on Social Structure:The introduction of human clones could disrupt the social fabric.It might lead to confusion in family relationships and could challenge the concept of individuality.6.Legal and Moral Dilemmas:There are no clear legal frameworks to deal with the rights and responsibilities of cloned individuals.This could lead to moral dilemmas and legal disputes.7.Economic Implications:The cost of cloning is currently very high,which could lead toa disparity in access,creating a divide between the rich and the poor who can afford such technologies.8.Biological and Genetic Diversity:Cloning could reduce genetic diversity,which is crucial for the survival and adaptability of the human species.A lack of diversity could make humanity more susceptible to diseases and environmental changes.9.Religious and Cultural Objections:Many religious and cultural beliefs oppose the idea of cloning,considering it to be against the natural order and divine will.10.Potential for Eugenics:Cloning could pave the way for a new form of eugenics, where only desirable traits are replicated,leading to a loss of diversity and potentialdiscrimination against those who are not clones.In conclusion,while cloning offers the potential for scientific advancements and potential medical benefits,the ethical,social,and psychological implications are profound and warrant serious consideration before proceeding with human cloning.It is essential to weigh the potential benefits against the risks and to establish robust ethical guidelines and legal frameworks to govern such practices.。
高中英语：unit2cloning课件 人教版选修8
Finally, the problem that occurred to the scientists was that a cloned animal lives __sh_o_r_te_r_th_a_n____ the original one.
Because__t_h_e__a_u_t_h_o_r__d_o_e_s__n_o_t__s_ta__te___ _a_n_y__p_e_r_s_o_n__a_l_o_p_i_n_i_o_n__a_b_o__u_t_c_l_o_n_i_n_g_.__
Task 4 Discussing and Speaking
3. 与同学讨论，共同解决问题。
Attention 注意：
切忌遇见生词就查字典，应该尽量根 据语境猜测词义。
Task 1 Main idea of each paragraph
__3____The problems of Dolly __2____ The major uses and procedures
as long as / shorter than / longer than
Para 4 The impact of Dolly
The word “controversial” in Para 4 probably means ____________. A. complicated B. arguable C. important
of cloning
__1____ The definition of cloning
高中英语 Unit 2 Cloning Section Ⅰ Warming Up Reading-
Unit 2 Cloning克隆实验开始于20世纪50年代，一步一个脚印的逐渐成熟。
那么，到底什么是克隆呢？如何进展克隆呢？WHAT IS CLONING?Have you ever wished you could have a clone of yourself to do your homework while you went out to play with your friends? Imagine if you could really do that, where would you start?What exactly is cloning?Cloning is a way of making an organism that is an exact genetic copy of another. This means that every single bit of DNA is the same between the two!You might not believe it, but there are human clones among us right now. They weren't made in a lab, though. They're identical twins, created naturally. Below, we'll see how natural identical twins relate to modern cloning technologies.How is cloning done?You may have first heard of cloning when Dolly the sheep showed up on the scene in 1997. Cloning technologies have been around for much longer than Dolly，though.How does one go about making an exact genetic copy of an organism? There are a couple of ways to do this: artificial embryo twinning and somatic cell nuclear transfer.How do these processes differ?1. Artificial Embryo TwinningArtificial embryo twinning is the relatively lowtech version of cloning. As the name suggests, this technology mimics the natural process of creating identical twins.2. Somatic Cell Nuclear TransferSomatic cell nuclear transfer (SCNT), uses an approach different from artificial embryo twinning, but it produces the same result: an exact clone, or a genetic copy, of another. This was the method used to create Dolly the sheep.1．organism n．有机体；生物体2．artificial adj. 人工的，人造的3．transfer v. 转移4．relatively adv. 相对地；比拟地5．mimic v. 模仿；模拟1．What is cloning?Cloning is a way of making an organism that is an exact genetic copy of another.2．What are the two ways of cloning mentioned in the passage?Artificial embryo twinning and somatic cell nuclear transfer.Section ⅠWarming Up & Reading－Preparing根据提示写出如下单词1．vi.不同，相异2．vt. 着手，从事，承当3．vt. 扔，投，掷4．vi. 反对，不赞成5．vt. 获得，赢得6．vt. 获得；到达(水平、年龄、状况等)7．vt. 禁止，不准8．vt.&vi. 积累，聚积9．adj. 准确的，准确的10．n. 突破【答案】 1.differ 2.undertake 3.cast 4.object5．obtain 6.attain 7.forbid 8.accumulate 9．exact 10.breakthrough看单词学构词：breakthrough为合成词，由break和through构成类似单词还有breakup“破裂〞；breakdown “失败，损坏，破裂〞等。
高中英语 Unit 2 Cloning What is cloning素材 新人教版选修8
高中英语 Unit 2 Cloning What is cloning素材新人教版选修8Cloning means the production of genetically identical animals. In 1995 scientists at the Roslin Institute in Edinburgh, Scotland produced Megan and Morag - two lambs cloned from a single embryo. In 1996 Dolly the sheep was produced, the first example of a clone produced not from an embryo cell but from a cell taken from an adult sheep. She was cloned using a nuclear transfer technique, where the nucl eus from a cell taken from the mammary gland of an adult sheep was fused with an un fertilized egg from which the nucleus had been removed. 277 cells were fused in this way, resulting in 29 viable embryos which were implanted into surrogate Blackface ewes. One gave birth to Dolly. Dolly died in 2003 after suffering the signs of p remature ageing such as arthritis and a liver complaint.The 'production' of Dolly has sparked a cloning frenzy. 1998 saw the arrival of cows cloned using the same technique that produced M egan and Morag 1. The Roslin institute cloned the first pig in 2000 2. In the US in 2001 a rhesus macaque monkey called Tetra was cl oned 3. Also in the US in 2002 a cloned kitten was reported 4 in the same year as the first cloned rabbits in France 5. In 2003 the first cloned mule 6, horse 7 and rat 8 were reported.Animal welfareCloning involves many possibilities for detrimental effects on animals. For example, cloned lambs and calves are frequently abnormally large at birth - some have been twice normal size. One cloning expert admitted in 1992 that "many cloned calves are abnormal".9 In Australia, a l amb of five times the mean birth weight for the breed has been recorded. In addition, cloned animals are often found to have malformed internal organs.10 Both of these adverse effects were seen in the first Edinburgh experiments in 1995 that led to Megan and Morag. In addition to death through malformed internal organs, one lamb had to be delivered by caesarean section because it had grown to twice its normal size in the womb, and all but one of the five cloned lambs were at least 20% larger than they should have been.Immediately after birth many cloned lambs have breathing difficulties, are lethargic and often will not suckle. As a consequence, perinatal mortality is high. Miscarriage rates are also hig h; this is thought to be at least in part because of 'large offspring syndrome'.11C loning techniques also require a great deal of surgical intervention. Those sheep destined to be egg donors undergo hormone injections, followed by surgery to have the egg cells removed. Following nuclear transfer, it is common for the cloned embryos to b e surgically implanted into temporary recipient sheep. Six days later, these temporary 'foster mothers' are simply killed and the embryos removed. The embryos are then placed, again surgically, into the surrogate mother ewes. Some deliveries have been achieved via Caesarean section. So the process is far from simple, and the surgery required can reasonably be expected to inflict residual pain. Attempts to create clones of animals that have been already cloned could also result in increased suffering. In France 1999, a calf which was a clone of a clone died aged eight weeks of rapid depletion of blood cells and severe anaemia. The researchers concluded "…somatic cloning may be the cause of long-term deleterious effects".12One possible application of cloning, suggested by the Roslin team, is the facilitation of farm animal genetic engineering. If successful, the cloning technique could be used to 'mass produce' farm animals which had been genetically engineered to grow faster, bigger or leaner. Cloning could also be used to mass produce animals genetically engineered to produce pharmaceutical protei ns in their milk or blood (gene pharming), or for use in xenotransplantation. However, genetic engineering itself has a very poor track record in terms of animal welfare, and the BUAV is appalled at the idea of combining genetic engineering and cloning techniques. Former Archbishop John Habgood has written of the motives behind cloning experiments:"But should science be going down this road at all? What is the point of it? The simple answer is - money. The driving force behind most of the research in this field has come from the agricultural industry. I use the word industry deliberately.Cloning is a means of standardising products, and that is what industry always wants." 13It is clear that cloning may cause a great deal of animal suffering. The so-called 'benefits', such as the production of genetically identical herds or flocks of animals for intensive farming, may be seen as dubious or even highly unethical. T he BUAV therefore believes that the practice of cloning should be stopped immediately.。
unit-2-cloning-Grammar课件
3. Teenagers shouldn’t spend too much time online. / Many Chinese parents hold the view.
Many Chinese parents hold the view that teenagers shouldn’t spend too much time online.
5. She was not al_t_o_g_e_th_e_r_ pleased at the corrections he made to her work.
6. Grey decided to move to the countryside and his wife made no o_b_j_e_ct_i_o_n_s to it.
4. Time travel is possible. / We have no scientific proof for the idea.
2 Replace the underlined parts with words that have the same meaning. Rewrite the sentence when necessary.
1. Compared with her family, and especially
3 Complete the passage using the correct form of the words below.
complicated fate attain cast accumulate reform carrier
As Freddy the frog grew older, he only had one ambition left (toa_t_ta_i_n___) – to continue his career forever. To do this he thought that he would have to clone himself. The doctor explained that this was a _c_o_m_p_l_i_ca_t_e_d__ procedure that he should consider carefully. Shortly after Freddy did some research, he discovered the _fa_t_e__ of Dolly the sheep. It seemed as if a clone was a _ca_r_r_i_e_r of more
克隆看法英语作文
克隆看法英语作文Title: The Cloning Debate: A Critical Examination。
Cloning, a topic that has sparked contentious debates worldwide, raises profound ethical, moral, and scientific considerations. Advocates tout its potential to revolutionize medicine and agriculture, while critics warn of ethical dilemmas and unforeseen consequences. In this essay, we delve into the multifaceted discourse surrounding cloning, examining both its promises and perils.Proponents of cloning argue that it holds immense promise for advancing medical research and treatment. Through therapeutic cloning, scientists aim to generate patient-specific stem cells for regenerative medicine, offering personalized therapies for conditions like Parkinson's disease and spinal cord injuries. Moreover, cloning could facilitate the production of organs for transplantation, addressing the critical shortage of donor organs and saving countless lives.In agriculture, cloning offers opportunities for enhancing crop yield, disease resistance, and livestock quality. By replicating superior plant or animal specimens, farmers can bolster food security and mitigate the impactsof climate change on agricultural productivity. Additionally, cloning endangered species presents alifeline for preserving biodiversity and restoring ecosystems on the brink of collapse.However, amidst these promises lie profound ethical concerns that demand careful consideration. The notion of cloning raises questions about the sanctity of life, individuality, and human dignity. Critics argue thatcloning undermines the inherent uniqueness of living beings, reducing them to mere commodities or objects of manipulation. Furthermore, the prospect of human reproductive cloning poses existential threats, challenging fundamental notions of parenthood, identity, and societal norms.Beyond ethical considerations, the scientificfeasibility and safety of cloning remain subject to scrutiny. Despite advancements in cloning techniques, the process still entails significant risks, including genetic abnormalities and health complications in cloned organisms. Moreover, the long-term implications of geneticmanipulation and cloning on biodiversity, ecosystem dynamics, and evolutionary trajectories remain poorly understood.Moreover, the commercialization of cloning raises concerns about equity, access, and socioeconomicdisparities. The commodification of cloned organisms could exacerbate existing inequalities, widening the gap between those who can afford such technologies and those who cannot. Additionally, the concentration of cloning technologies in the hands of a few corporations or nations may further marginalize developing countries and perpetuate globalpower imbalances.In light of these complexities, a nuanced approach to cloning is imperative, one that balances scientificprogress with ethical considerations and societal values.Regulation and oversight mechanisms must be robustly enforced to ensure responsible research practices and safeguard against potential abuses. Transparency, public engagement, and interdisciplinary dialogue are essentialfor fostering informed decision-making and navigating the ethical dilemmas inherent in cloning.Furthermore, investments in alternative approaches, such as induced pluripotent stem cells and gene editing technologies, offer promising avenues for achieving therapeutic goals without the ethical and technical challenges associated with cloning. By diversifying research efforts and exploring complementary methodologies, we can harness the transformative potential of biotechnology while upholding ethical principles and human dignity.In conclusion, the cloning debate embodies theintricate interplay between science, ethics, and society. While cloning holds promise for advancing medicine, agriculture, and conservation, it also raises profound ethical concerns and uncertainties. As we navigate thisfrontier of biotechnology, we must approach cloning with caution, humility, and a steadfast commitment to ethical principles, ensuring that scientific progress serves the greater good of humanity while respecting the intrinsic value of life.。