構(gòu)建不同質(zhì)粒評(píng)估抗質(zhì)粒接合作用(英文版).docx

ZHENG CONSTRUCTION OF REPORTER PLASMIDS FOR THE EVALUATION OF NATURAL PRODUCTS AS ANTI-PLASMID AGENTSCONSTRUCTION OF REPORTER PLASMIDS FOR THE EVALUATION OF NATURAL PRODUCTS AS ANTI-PLASMID AGENTSThis dissertation is submitted in part fulfilment of the requirements for the Master in Pharmacognosy, Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, University College London.Candidate Name:Zheng ZhejiaoCandidate Number:14089435Supervisor: Dr. Paul StapletonDate:28/8/2015Word Count: 9430Issue Date: 24/08/2015 Page 55 of 55 Revision: B PLAGIARISM STATEMENTThis thesis describes research conducted in the School of Pharmacy, University College London between May 2015 and August 2015 under the supervision of Dr. Paul Stapleton. I certify that the research described in this work is original and that any parts of the work that I have been conducted in collaboration are clearly indicated. I also certify that I have written all the text herein and have clearly indicated by suitable citation any part of this dissertation that has already appeared in publication.Signed in London Friday, August 28, 2015: (Signature & Candidates Name in Print) Zheng ZhejiaoACKNOWLEDGMENTSThanks to Dr. Paul Stapleton, who provided me the topic of the project. He also gave me some instructions during the experiments and dissertation writing. Thanks to Mr. Pedro Ernesto de Resende and Miss Sarah Soares, who taught me the skill of the experiments and helped me to finish them. Also, thanks to all other staff in the laboratory.TABLE OF CONTENTSContents1.Introduction111.1The relationship between Plasmid conjugation and antibiotics resistence111.2Bioluminescence and Lux operon141.3Reporter strains to study bioactivities including anti-conjugation assay162.Aims of Project183.Material and Methods193.1 The Strains193.2 Reagents203.3 Equipment213.4 Method213.4.1 Assay 1: Setting up the anti-conjugation assays and comparison of two different methods of agar preparing in wells213.4.2 Assay 2: Test the influence of Tnlux and lacI in pKM101 on the conjugation of JM109244.Results274.1 The Results of Assay 1: Setting up the anti-conjugation assays and comparison of two different methods of agar preparing in wells274.2 The Result of Assay 2: Test the influence of Tnlux and lacI in pKM101 on the conjugation of JM109305.Discussion335.1 Assay 1: setting up the anti-conjugation assays and comparison of two different methods of agar preparing in wells335.1.1 The anti-conjugation activity of BiTC and its solvent DMSO335.1.2 The differences between De La Cruzs and Sarahs method345.1.3 The theories in the De La Cruzs article about the light emission conjugation assay375.2 Assay 2: Test the influence of Tnlux and lacIq in pKM101 on the conjugation of JM109395.2.1 The influence of Tnlux and lacIq insertion395.2.2 The variation in Assay 2416.Conclusions and Future Work45Appendix 1: The conjugation transfer frequency of 4 different groups of donors and recipients477.References50TABLE OF FIGURESFigure 1. 1 The Structure of Plasmid pKM101 1213Figure 1. 2 The Process of Conjugation 14Figure 1. 3 The Structure of Lux Operon 16Figure 3. 1 The Strains Labelled with Lux and Lacl used in the Project20Figure 3. 2 The Structure of the Plasmid pBCSKThe20Figure 3. 3 The Marks of Agar Plates in CFU Counting24Figure 4. 1 The Luminescence of Donor + Recipient of Two Methods in BiTC and DMSO28Figure 4. 2 The Comparison of Plasmid Conjugation Transfer Frequency in JM109 (pKM101:Tnlux22+pBCSK:laclq) & ER1793(F-) between BiTC Groups and DMSO Groups28Figure 4. 3 The Logarithm of Luminescence of ER1793 (pKM101lux22) of Two Methods in BiCT and DMSO30Figure 4. 4 The Luminescence of ER1793 (pKM101lux22) of Two Methods in BiCT and DMSO30Figure 4. 5 Plasmid Transfer Frequency of Labelled and Unlabelled pKM101 from E.coli JM109 to E.coli ER179332Figure 5. 1 The Chemical Structure of BiTC33Figure 5. 2 The Regression of Transconjugants and Luminescence in Assay 138Figure 5. 3 The Sequences of the Lac Operon 5440Figure 5. 4 The Relationship with Plate Counting and Numbers of Replicates in Accuracy 5944TABLE OF TABLESTable 3. 1 The Cells and Compounds Added in Assay 123Table 3. 2 Table The Strains Added in the Microtitre Plates in Assay 226Table 5. 1 The Comparison of RSD% in BiTC Groups in De La Cruzs and Sarahs Method35Table 5. 2 The Light Production and Light Inhibition38Table 5. 3 The Original Data of One of Groups42ABBREVIATIONSBiTC = benzyl isothiocyanate DMSO = Dimethyl sulfoxideLB = Lysogeny brothPBS = Phosphate buffered salineRSD = relative standard deviation CFU = colony-forming unit AHL= N-acyl l-homoserine lactones ABSTRACTObjectives: The aim of the project was to construct plasmids with different strains, and set up the assays to test the anti-conjugation antivity of natural products by labelling with lux operon to detect their luminescence. Also, the project studied the factors influencing the anti-conjugation assay such as different agar preparation methods and inserted gene.Materials and Methods: There were 2 assays included in the project. The first assay was to set up anti-conjugation assay by labelling pKM101 with Tnlux gene. Also, the different agar preparation methods were compared. One was to add agar and BiTC or DMSO to each well separately. Then they were kept in the fridge for overnight. (Essays) Another one was to mix BiTC or DMSO in the agar, then they were pipetted into each well, which were kept in the fridge for 10 minutes (Sarahs). The second assay was to detect influence of Tnlux and lacIq by labelling JM109 (pKM101) with Tnlux and pBCSK:lacIq.Key findings: In the Assay 1, BiTC had high inhibition in both donors and recipients groups and contrast groups. According to De La Cruzs theory, BiTC had no anti-conjugation activity due to have over 50% inhibition in contrast groups. For comparing, in BiTC groups, Sarahs data were much lower than Essays one, but higher in DMSO groups. Conjugation frequency was higher when JM109 were labelled with Tnlux, and had some changes when they were labelled with pBCSK:lacIq.Conclusion: The anti-conjugation assay has been set up based on labelling with lux to detect luminescence and inhibition on both test and constrast groups and the method is reproducible. Tnlux might improve the anti-conjugative activity. LacIq and other part of pBCSK also might influence the anti-conjugative activity, which cannot be ignored in the assay. Keywords: Antibiotics resistance, Plasmid conjugation, Lux operon, Report gene, Benzyl isothiocyanate, Lac operon1. INTRODUCTION1.1 THE RELATIONSHIP BETWEEN PLASMID CONJUGATION AND ANTIBIOTICS RESISTENCESince the first discovery of penicillin due to the contamination of Staphylococcus by a kind of fungi by Alexander Fleming in 1928, people started to use antibiotics to treat infectious diseases. The history learning site, C N Trueman (2015) Antibiotics http:/www.historylearningsite.co.uk/a-history-of-medicine/antibiotics/ Accessed: 01/07/2015 In the following century, more and more antibiotics were discovered, such as in -lactams, nitrofurans, aminoglycosides, chloramphenicol, tetracyclines (These antibiotics were introduced between 1940s and 1960s) and so on. M. A. Fischbach, C. T. Walsh (2009) Antibiotics For Emerging Pathogens Science 325(2009):1089-1093 Antibiotics have been used to treat a variety of diseases with infection, diphtheria, whooping cough and tuberculosis, BBC (2014) Antibiotics and immunisation http:/www.bbc.co.uk/bitesize/ks3/science/organisms_behaviour_health/disease/revision/6/ Accessed: 24/7/2015 including pneumonia, which was fatal in the past centuries. Osler, William (1901). Principles and Practice of Medicine, 4th Edition. New York: D. Appleton and Company. p. 108. However, with the increasing using of antibiotics, here comes a problem that there are more and more antibiotics resistances. Antibiotics resistance is a kind of phenomenon that some species of microbial can resist new drugs that deal with them by evolution, so that these antimicrobial reagents no longer have effects on these species. J. ONeill (2014) Antimicrobial Resistance: Tackling a crisis for the health and wealth of nations The Review on Antimicrobial Resistance Recently, a type of bacterial which contains the strains of NDM-1 has appeared with resistance of most antibiotics and antimicrobials used by people, which will deteriorate the health of human being. H. Qua, X. Wang, Y. Ni, J. Liu, R. Tan, J. Huang, L. Li, J. Sun (2015) NDM-1-producing Enterobacteriaceae in a teaching hospital in Shanghai, China: IncX3-type plasmids may contribute to the dissemination of blaNDM-1 International Journal of Infectious Diseases 34(2015): 8-13 There are several mechanisms which can result in the antibiotic resistance, such as efflux pump, changing targets and antibiotics and preventing the drug from entering the cell. M. Lin, Y. Lin, C. Tu, C. Lan (2015) Distribution of different efflux pump genes in clinical isolates of multidrug-resistant Acinetobacter baumannii and their correlation with antimicrobial resistance Journal of Microbiology, Immunology and Infection Available online 14 May 2015 CM. Armstrong, DJ. Meyers, LS. Imlay, CF. Meyers, AR. Odom (2015) Resistance to the antimicrobial agent fosmidomycin and an FR900098 prodrug through mutations in deoxyxylulose phosphate reductoisomerase (Dxr). Antimicrob Agents Chemother AAC.00602-15 Some of these mechanisms are resulted from mutation, which can alter their ability to resist antibiotics. E. Ibargen-Mondragn, S. Mosquera, M. Cern, E.M Burbano-Rosero, S.P. Hidalgo-Bonilla, L. Esteva, J. P. Romero-Leitn (2014) Mathematical modeling on bacterial resistance to multiple antibiotics caused by spontaneous mutations Biosystems 117(2014): 60-67 Others are from the antibiotic resistance gene of other bacteria. Among of these, plasmids play an important role on gaining these resistance gene and DNA for bacteria from outside. D.B Clewell (2001) Antibiotic Resistance Plasmids in Bacteria John Wiley & Sons, Ltd Plasmids are kind of circular DNA molecule which are different from the chromosomal DNA of bacteria. They contains origin of replication (ori) and a lot of genes, including drug resistance gene, so they can replicate by using the chromosomal DNA and survive in certain environment. D. Summers (2009) The Biology of Plasmids John Wiley & Sons PM Bennett (2008) Plasmid encode antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria British Journal of Pharmacology 153(2008):S347-S357 Fig.1.1 shows the structure of pKM101, which is a 35.4 kb plasmid from plasmid R46. In pKM101, the gene including bla (for ampicillin resistance), muc (for resistance of UV mutagenesis) and tra (for conjugation) P J Langer, W G Shanabruch, G C Walker (1981) Functional organization of plasmid pKM101 J Bacteriol 145(1981): 1310-1316. The plasmids can also be divided into several types, such as R plasmid (resistance plasmid), which contains resistance gene to resist one or several antibiotics and antimicrobial reagents, and F plasmid (fertility plasmid), which is relevant to sex pilus and responsible for conjugation. K. Hardy, Kimber G. Hardy (1981) bacterial plasmids Wokingham: Van Nostrand Reinhold(UK) Co.Ltd Conjugation is a kind of plasmid mobility that DNA strand is transferred from one cell to another. C. Smillie,M. P. Garcillan-Barcia, M. V. Francia, E. P. C. Rocha, F. de la Cruz (2010) Mobility of Plasmids MICROBIOLOGY AND MOLECULAR BIOLOGY 74(2010): 434-452 In general, there are various conjugation processes in different types of plasmids, and here use F plasmid as an example, which has conjugation system via sex pilus. Bacteria cell is regarded as donor (F+) when with F plasmid. On the other hand, it is recipient (F-) when without F plasmid. When donors and recipients meet together, they will be connected by sex pilus, so that one strand DNA of F plasmid can transfer to another cell through sex pilus and replicate, thus another F plasmid forms in the donor cell. M.J. Pelczar (2010) Microbiology:Application Based Approach Tata McGraw Hill Education Private Limited The transconjugants are defined as the result of donors and recipients mating. M. Tariq, M. R. Lum, A. W. Chong, A. B. Amirapu, S. Hameed, A. M. Hirsch (2015) A Reliable Method for the Selection and Confirmation of Transconjugants of Plant Growth-Promoting Bacteria especiallyPlant-Associated Burkholderia spp. Journal of Microbiological Methods Available online 14 July 2015 Fig.1.2 shows the process of conjugation. iGEM 07 (2007) Boston University/Conjugation http:/2007./wiki/index.php/Boston_University/Conjugation Accessed: 04/07/2015Apart from F plasmid, other kinds of plasmids such as R plasmid also have activities of conjugation. H Kruse and H Srum (1994) Transfer of multiple drug resistance plasmids between bacteria of diverse origins in natural microenvironments. Appl Environ Microbiol. 60(1994): 4015-4021 During the process of conjugation, a lot of antibiotic resistance genes are transferred. Repeating such process, with the time increasing, there are various drug resistance gene in the plasmid, which will develop different resistances in the environment of antibiotics. So that plasmid conjugation is one of the most important factors of increasing of muti-drug resistance.11 Figure 1. 1 The Structure of Plasmid pKM101 12Figure 1. 2 The Process of Conjugation 171.2 BIOLUMINESCENCE AND LUX OPERONIn the field of biology, there is a phenomenon that some creatures will produce light by themselves, which can be called bioluminescence. There are a lot of species can produce light, such as jellyfish, fireflies, krill and some bacteria including V. fischeri, V. harveyi and P. leiognathi. T. Wilson (2013) Bioluminescence: Living Lights, Lights for Living Harvard College E. A Meighen (1991) Molecular biology of bacterial bioluminescence. Microbiol Rev. 55(1991): 123-142 In natural system, these species use bioluminescence for communication, searching for food, defence, mating and so on. Tracy V. Wilson, Howstuffworks (2015) How Bioluminescence Works /anim al-facts/bioluminescence2.htm Accessed: 05/07/2015 The mechanisms of bioluminescence vary from specie to specie, but mostly, including bacteria and fireflies, are through luciferase. For fireflies, it is a luciferin-luciferase system. The luminescence of fireflies occurs in a special organ which is named as lantern. The D-luciferin was oxidized to oxyluciferin and the light was produced. M. S. Waidmann, F. S. Bleichrodt, T. Laslo , C. U. Riedel (2011) Bacterial luciferase reporters: The Swiss army knife of molecular biology Bioengineered Bugs 2(2011): 8-16 In bacteria, it is a bit similar way. However, rather than luciferin, the substance oxidized in luminescence bacterial is flavin mononucleotide (FMNH2). The produce of the reaction is like following. First of all, fatty acid reductase complex catalyses the fat to fatty acid. Then, the fatty acid is reduced to aldehydes via ATP, NADPH and reductase complex. On the other hand, NADPH flavin reductases may catalyse the production of FMNH2. However, the reation of this step is unsure. 21At last, the aldehydes react with FMNH2 under the catalysis of bacterial luciferase, so that the FMN forms and light is produced at last. The equation of the reaction is like this: J. W. Eckstein , J. W. Hastings , S. Ghisla (1993) Mechanism of bacterial bioluminescence: 4a,5-Dihydroflavin analogs as models for luciferase hydroperoxide intermediates and the effect of substituents at the 8-position of flavin on luciferase kinetics Biochemistry 32(1993): 404-411 RCOX+HOHfatty acid reductaseRCOOH+XHRCOOH+ATP+NADPHfatty acid reductaseNADP+AMP+PPi+RCHOFMNH2+RCHO+O2LuciferaseFMN+RCOOH+H2O+lightLux operon is the gene sequence which give these bacteria the characteristic of luminescence, which consists of CDABE. Among of these genes, lux AB have the relationship with luciferase while lux CD and lux E are for fatty acid reductase. In some species, there is also lux G which encodes flavin reductases. In photobacterium species, there is also a lux F in the lux gene. However, there is no use in luminescence in bacteria. In V.harveyi, there is a lux H gene, which possibly participate in the synthesis of FMN, which is the part of luminescence reaction as well.23 Fig.1.3 shows the sequence of lux operon in some species. The bioluminescence can be applied widely in technologies. For example, in biosensors, lux gene is introduced to track the analytes wanted. Lux gene is used in imaging to reveal the tissues and cells to be analysed. Lux gene also can be used in labelling for immunoassays. Among of these, lux gene is used as reporter gene, which makes the detection more sensitive and convenient. A. Roda , P. Pasini, M. Mirasoli, E. Michelini, M. Guardigli (2004) Biotechnological applications of bioluminescence and chemiluminescence Trends in biotechnology 22(2004): 295-303Figure 1. 3 The Structure of Lux Operon 221.3 REPORTER STRAINS TO STUDY BIOACTIVITIES INCLUDING ANTI-CONJUGATION ASSAYDue to the increasing antibiotic resistance and less novel compounds to be found since the introduction of antibiotics in treating infecting diseases, scientists start to focus their antimicrobial reage