VRIJE UNIVERSITEIT BRUSSEL        INSTITUTE OF MOLECULAR BIOLOGY AND                   BIOTECHNOLOGYTITLE: VIROLOGY PRACTI...
LIST OF TABLESTable 1 Results of plaques on indicator bacteria ..............................................................
Table of ContentsSpontaneous induction of Bacteriophages.....................................................................
Figure 3: Results of Agarose gel of phage DNA................................................................................
Spontaneous induction of Bacteriophages1.1 IntroductionBacteriophages follow either a lytic or lysogenic life cycles when ...
Bacteriophage λ and its relatives are temperate phages, this means that they can show bothlysogenic and lytic lifestyles. ...
2.3 Results and discussionTable 2. Number of plaques observed after phage culture in two host bacteria                    ...
Restriction and modification of bacteriophage λ in different E.coli strains.3.1 IntroductionRestriction modification syste...
Table 4: The day 3 results showing the plaques after culture on the three bacterial strains                       λK514   ...
When λHB101 was introduced in K514 it showed an efficiency of plating of 1 which shows thatit does not have the restrictio...
Plagues were observed in petri dishes with bacteria strains C600 and C600(λimm34) but not inC600λ. This is because the lam...
Table 8: Relative distances and Molecular weight tabulation of bacteria Stx geneMigration    Distance Relative Distance   ...
Molecular weight = 2818bp5.2.2 Results of phage DNA1λ 2ψ     3c   4   5   6      7   8   9   10     11     12   PstFigure ...
Sample Relative Distance = 0.575                               4.5                                                        ...
belongs to class II of transposable element with an accessory determinant of antibiotic resistancein addition to transposi...
After incubating 100 colonies overnight in MinA plates supplemented with different amino acidmixtures and rich LB media re...
a) 0.1 ml MG1655 + 0.2 ml of P1 vir lysate    b) 0.1 ml MG1655 + 0.2 ml of P1 vir lysate    c) 0.1 ml MG1655 + 100µl LB wi...
Figure 5: Agglutination was not observed when the bacteria and yeast were incubated oniceReferencesAtsumi S. and JW Little...
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Virology practical report

  1. 1. VRIJE UNIVERSITEIT BRUSSEL INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGYTITLE: VIROLOGY PRACTICALS REPORTNAMES: KARIUKI SAMUEL MUNDIAINSTRUCTOR: Prof. Dr. Henri De GreveASSISTANCE: Maia De Kerpel and Cine DeboeckDATE OF SUBMISSION: 27/04/2012 1
  2. 2. LIST OF TABLESTable 1 Results of plaques on indicator bacteria ............................................................................ 5Table 2. Number of plaques observed after phage culture in two host bacteria ............................. 7Table 3 Day 2 phage results showing the plaques after incubation with bacteriophage lambdaovernight. ........................................................................................................................................ 8Table 4: The day 3 results showing the plaques after culture on the three bacterial strains........... 9Table 5 showing efficiency of plating ............................................................................................ 9Table 6: showing phenotypes of the three bacterial strains .......................................................... 10Table 7: Results of phage immunity test....................................................................................... 10Table 8: Relative distances and Molecular weight tabulation of bacteria Stx gene ..................... 12Table 9: Relative distances and molecular weight tabulation of phage Stx gene ......................... 13Table 10: Number of bacterial colonies after incubation in LB and Mackonkey media .............. 15Table 11: Results in MinA plates supplemented with different amino acids showing cysteinepositive plates................................................................................................................................ 15LIST OF FIGURESFigure 1 showing results of Agarose gel of total bacterial DNA.................................................. 11Figure 2: Graph of log of molecular weight and relative distance of band migration.................. 12Figure 3: Results of Agarose gel of phage DNA .......................................................................... 13Figure 4: Graph of log of molecular weight and relative distance of band migration .................. 14Figure 5: Agglutination was not observed when the bacteria and yeast were incubated on ice ... 18 2
  3. 3. Table of ContentsSpontaneous induction of Bacteriophages................................................................................................... 5 1.1 Introduction ........................................................................................................................................ 5 1.2 Objective: ............................................................................................................................................ 5 1.3 Results and discussion ........................................................................................................................ 5 1.3.1 Results.......................................................................................................................................... 5 Table 1 Results of plaques on indicator bacteria.................................................................................. 5Suppression of amber mutations.................................................................................................................. 6 2.1 Introduction ........................................................................................................................................ 6 2.2 Objective ............................................................................................................................................. 6 2.3 Results and discussion ........................................................................................................................ 7Table 2. Number of plaques observed after phage culture in two host bacteria......................................... 7Restriction and modification of bacteriophage λ in different E.coli strains. ................................................ 8 3.1 Introduction ........................................................................................................................................ 8 3.2 Objective ............................................................................................................................................. 8 3.3 Results and discussion ........................................................................................................................ 8Table 3 Day 2 phage results showing the plaques after incubation with bacteriophage lambda overnight....................................................................................................................................................................... 8Table 4: The day 3 results showing the plaques after culture on the three bacterial strains ...................... 9Table 5 showing efficiency of plating............................................................................................................ 9Table 6: showing phenotypes of the three bacterial strains ...................................................................... 10Immunity of lysogenic bacteria...................................................................................................................10 4.1 Introduction ...................................................................................................................................... 10 4.2 Objective ........................................................................................................................................... 10 4.3 Results and discussion ......................................................................................................................10Table 7: Results of phage immunity test. ................................................................................................... 10Isolation of total bacterial DNA and phage DNA and PCR amplification of shiga toxin gene.....................11 5.1 Objective ........................................................................................................................................... 11 5.2.1 Results of total bacterial DNA.................................................................................................... 11Table 8: Relative distances and Molecular weight tabulation of bacteria Stx gene ................................... 12Figure 2: Graph of log of molecular weight and relative distance of band migration................................12 5.2.2 Results of phage DNA.................................................................................................................13 3
  4. 4. Figure 3: Results of Agarose gel of phage DNA...........................................................................................13Table 9: Relative distances and molecular weight tabulation of phage Stx gene ...................................... 13Figure 4: Graph of log of molecular weight and relative distance of band migration................................14Transposition of TN10 from phage λ vehicles.............................................................................................14 6.1 Introduction ...................................................................................................................................... 14 6.2 Objective ........................................................................................................................................... 15 6.3.1 Results and Discussion ...............................................................................................................15Table 10: Number of bacterial colonies after incubation in LB and Mackonkey media.............................15 6.3.2 Results isolation and identification of amino acid biosynthesis mutants.................................. 15Table 11: Results in MinA plates supplemented with different amino acids showing cysteine positiveplates...........................................................................................................................................................15Generalized Transduction: P1 Transduction...............................................................................................16 7.1 Introduction ...................................................................................................................................... 16 7.2 Objective ........................................................................................................................................... 16 7.3 Results and Discussion ......................................................................................................................16Yeast agglutination ..................................................................................................................................... 17 8.1 Introduction ...................................................................................................................................... 17 8.2 Objective ........................................................................................................................................... 17 8.3 Results and Discussion ......................................................................................................................17Figure 5: Agglutination was not observed when the bacteria and yeast were incubated on ice .............. 18References ..................................................................................................................................................18 4
  5. 5. Spontaneous induction of Bacteriophages1.1 IntroductionBacteriophages follow either a lytic or lysogenic life cycles when they infect a bacteria cell.Lysogeny is obtained when a bacteriophage DNA is integrated in the host genome, a conditionthat arises during adverse conditions of the environment, rise in virus population or physiologicalchanges of the bacteria cell. Phage induction occur when host DNA is damaged or heat treatmentof temperature sensitive repressor protein. However, one million out of one billion bacterial cellscontaining prophage can undergo spontaneous induction and revert to lytic type of lifestyle. Thisis due to activation of the genes that enhances the lytic life cycle.1.2 Objective:To isolate viable phages from lysogenic bacterial strains and illustrating it by using indicatorEscherichia coli strains.1.3 Results and discussion1.3.1 ResultsTable 1 Results of plaques on indicator bacteria 100 101λ plaque plaqueΨ933 plaque No plaqueLysis of the indicator E.coli C was observed in both the 100 and 101 dilutions of bacteriophage λand in 100 of ψ933 but not in 101 dilution of ψ933 phage. However, more complete confluentplaque was observed in 100 dilution as compared to 10-1 dilution of λ phage. In addition λ hadmore complete confluent plaque compared to ψ933.Since spontaneous induction is a radomn chance event, we expected to observe plaques if itreally happened. The observation of plaques shows that the phage DNA was excised from thebacterial genome and followed the lytic lifestyle. 5
  6. 6. Bacteriophage λ and its relatives are temperate phages, this means that they can show bothlysogenic and lytic lifestyles. In lysogeny, the phage DNA integrates into a very specific regionof the bacteria genome called attB using its attP, a process catalyzed by λ Int protein and theIntegration host factor (IHF) from E.coli host. This idea of integration of the λ genome into thehost chromosome by a process of reciprocal crossing over was first proposed by Allan Campellin 1962. The reversal of this process, excision of the prophage, requires besides Int and IHF alsothe λ Xis protein and occurs by recombination between the attL and attR sites.The results showed that spontaneous induction was possible without use of any inducingsubstance such as UV light or chemicals.Suppression of amber mutations2.1 IntroductionAmber mutation is a change in nucleotide sequence from a codon that codes an amino acid toUAG stop codon leading to production of non-functional protein since UAG when on amessenger RNA is a signal for termination of translation. Most codons on messenger RNA codefor amino acids which are gradually added to the growing polypeptide chain which eventuallyleads to production of functional proteins. Stop codons on the other hand bind release factorleading to the dissociation of the two ribosomal subunits and consequence termination oftranslation. Other stop codons in RNA are opal (TGA) and ochre (TAA).Bacteriophages with amber mutations are able to grow in bacteria cells that have a mutant tRNAcalled amber suppressors. These bacteria’s tRNA is able to read through UAG and produce afunctional protein. This replaces the original functional protein of the wild type virus hence thesebacteria are called amber suppressor mutants.2.2 ObjectiveTo observe the results of culturing λ1098 bacteriophage carrying Tn10tet transposon insuppressor minus (Su-) and suppressor plus (Su+) host. 6
  7. 7. 2.3 Results and discussionTable 2. Number of plaques observed after phage culture in two host bacteria 107 DILUTION FACTORCSH 110 25 PLAQUESC600 NO PLAQUESThere were no plaques observed in the plate with. E.coli C while 25 plaques were observed on107 dilution and more confluent plaques on other dilutions in the petri dish with E.coli CSH110strain.These results reveal that the E.coli CSH110 strain is suppressing the amber mutations of thederivative phage. In contrast, E.coli C600 is a wild type isolate which does not suppress theamber mutations of the λ1098 phage.The presence of amber suppressor in CSH110 strain enabled the protein synthesis to occurnormally despite the presence of amber mutations. This allowed the replication of the phagegenome with eventual lysis of bacteria which produced visible colonies on the plates. On theother hand, strain C did not display the phenotype under permissive conditions.Amber Suppressors are mutant tRNA genes that code for tRNA whose anticodons have beenaltered so that they respond to UAG (amber) codons and insert the amino acid of the wild typetRNA instead of release factors.The titre of the phage stock is calculated as follows:As indicated in the above table, 25 plaques were observed on Su+CSH110 petri dish in the 10-7dilution. Therefore, the 10-7 dilution tube contains 25PFU per 0.02ml, and the titre of the virus inthe stock is 25/0.02x107 PFU/ml or 1.25x1010 PFU/ml. 7
  8. 8. Restriction and modification of bacteriophage λ in different E.coli strains.3.1 IntroductionRestriction modification system is used to protect bacteria from incoming foreign DNA from theenvironment including bacteriophages at the same time modify its DNA to prevent restriction ofits chromosome from this nuclease activity. This system has been observed to prevent growth ofbacteriophages from other bacterial host. Restriction is realized by use of sequence specificendonucleases while modification it majorly by methylation of these specific sequences.Restriction modification systems are classified as type I, type II and type III depending on theorder of their discovery. Type II restriction modification systems are the major one used in thelabs in genetic engineering because they are site-specific. Type I system is not site-specific and isthe major system in E.coli K12.3.2 ObjectiveTo illustrate the phenomenon of restriction and modification of DNA using bacteriophage λ as amodel.3.3 Results and discussionTable 3 Day 2 phage results showing the plaques after incubation with bacteriophagelambda overnight. Plaques at 107 dilution Stock titreK514 20 1x1010C600 48 2.4x1010HB101 21 1.05x1010The 10-7 dilution factor showed clearly countable plaques in all the three E.coli K12 strains asindicated in the table above. Each mini-lysate was titrated after dilutions on the three E.colistrains again and the results were as follows. 8
  9. 9. Table 4: The day 3 results showing the plaques after culture on the three bacterial strains λK514 λC600 λHB101K514 27 = 1.35x109pfu/ml confluent confluentC600 confluent 34 = 1.7x109pfu/ml 17 = 8.5x104pfu/mlHB101 confluent confluent 24 = 1.2x109pfu/mlTable 5 showing efficiency of platingOriginal strain titre (pfu/ml) New strain titre (pfu/ml) Efficiency of plating (no units)K514 = 1.0x1010 K514 = 1 C600 = 1 HB101 = 1C600 = 2.4x1010 K514 = 1 C600 = 1 HB101 = 8.5x104 3.5x10-6HB101 = 1.05x1010 K514 = 1 C600 = 1 HB101 = 1The efficiency of plating reduced by 10-6 when λHB101 was grown in C600 bacterial strain plateand only a small number of bacteria produced progeny phages. This clearly confirms that C600has a restriction and modification system since every bacterium with a restriction system musthave a modification system to protect its DNA from degradation by nucleases. The titre reducedafter introduction of λHB101 in C600 bacterial strain due to degradation of the phage DNA bythe restriction enzymes of the bacterial host. It is clear here that bacterial strain HB101 does nothave a modification system otherwise it could have given the progeny phage the modification tosurvive C600 restriction. Consequently, it cannot have a restriction system as well, otherwise itwould destroy its own DNA.However, some of the phage DNA was able to survive due toacquiring the modification by methylation of the restriction site by C600 as it modified its DNA. 9
  10. 10. When λHB101 was introduced in K514 it showed an efficiency of plating of 1 which shows thatit does not have the restriction capability as shown by C600 strain against the same phage.Finally, when λK514 was introduced in C600 it showed an EOP of 1. This implies that it had amodification system which prevented degradation of its incoming phage unlike the case ofλHB101. The conclusion is summarized in the table below.Table 6: showing phenotypes of the three bacterial strains Restriction ModificationK514 - +C600 + +HB101 - -Immunity of lysogenic bacteria4.1 IntroductionBacterial phages in symbiosis or lysogeny with host bacteria give immunity to that host againstlysis by consequence infection by homologous free phages. This implies that lambda phageparticipates in its own interference. It does so by the prophage coding for a diffusible protein thatis involved in this interference by blocking the expression of the incoming bacteriophagegenome.4.2 ObjectiveTo illustrate the phenomenon of bacteriophage immunity, using bacteriophage lambda as amodel.4.3 Results and discussionTable 7: Results of phage immunity test.Bacteriophage ObservationC600 PlaquesC600(λ) No PlaquesC600(λimm34) Plaques 10
  11. 11. Plagues were observed in petri dishes with bacteria strains C600 and C600(λimm34) but not inC600λ. This is because the lambda prophage already in C600(λ) prevented expression of thegenome of incoming homologous phage. It does so by producing a repressor protein that blocksthe expression. C600 is a wild type and therefore does not contain the lambda prophage whileC600(λimm34) has a mutant lambda phage giving both of the ability to lyse the bacteria since therepressor protein works on a homologous phage.The resident lambda phage produces a cI repressor protein that binds to the O L and OR sites ofthe super infecting lambda phage as soon as it enters the cell turning off the expression of theincoming phage gene and hence no plaques result.Isolation of total bacterial DNA and phage DNA and PCR amplification of shiga toxin gene5.1 ObjectiveTo isolate bacterial total DNA, perform PCR and calculate its molecular weight of the shigatoxin genes.5.2.1 Results of total bacterial DNATotal bacterial DNA was obtained and PCR amplification of shiga toxin performed and resultswere as below. 1λ 2ψ 3c 4 5 6 7 8 9 10 11 12 13 14 15 Pst Figure 1 showing results of Agarose gel of total bacterial DNA 11
  12. 12. Table 8: Relative distances and Molecular weight tabulation of bacteria Stx geneMigration Distance Relative Distance Molecular weight (bp) Log molecular weight(cm)0.9 0.24 11501 4.061.1 0.29 5077 3.711.6 0.42 2838 3.451.9 0.50 2140 3.332.1 0.55 1986 3.302.4 0.63 1700 3.23Dye front 3.8cm 4.5 y = -1.8146x + 4.3344 4 R² = 0.8695 3.5 Log of molecular weight 3 2.5 2 1.5 1 0.5 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Relative distance of band migrationFigure 2: Graph of log of molecular weight and relative distance of band migrationSample relative distance = 0.47Equation of line y = -1.8146x+4.3344Substitution = -1.8146(0.47)+4.3624Antilog = 3.4 12
  13. 13. Molecular weight = 2818bp5.2.2 Results of phage DNA1λ 2ψ 3c 4 5 6 7 8 9 10 11 12 PstFigure 3: Results of Agarose gel of phage DNATable 9: Relative distances and molecular weight tabulation of phage Stx geneMigration Distance Relative Distance Molecular weight (bp) Log molecular weight(cm)1.2 0.30 11501 4.061.6 0.40 5077 3.712.2 0.58 2838 3.452.5 0.63 2140 3.332.6 0.65 1986 3.303.1 0.78 1700 3.23Dye front = 4cm 13
  14. 14. Sample Relative Distance = 0.575 4.5 y = -1.7377x + 4.4807 4 R² = 0.934 3.5 log of molecular weight 3 2.5 2 1.5 1 0.5 0 0 0.2 0.4 0.6 0.8 1 Relative distance of band migrationFigure 4: Graph of log of molecular weight and relative distance of band migrationEquation = y= -1.7377x + 4.4807Substitution = x = 0.575Y=-1.7377 (0.575) + 4.4807Molecular weight = 2960bp.The PCR analysis showed a visible band at the 933W phage and not at the λ or control well. Thisconfirmed that the shiga toxin was gene was present in the 933W phage and after integreationinto the bacteria its presence could be located as well by use of forward and reverse PCR primersspecific for it.In addition using Pst marker with known molecular weight it was possible to confirm themolecular weight of our shiga toxin gene which was approximately 2930 base pairs.Transposition of TN10 from phage λ vehicles6.1 IntroductionTransposable elements are discrete DNA segments that can repeatedly be inserted in the hostgenome. The process occurs without DNA sequence homology as contrasted to DNArecombination. Kleckner, 1981 has classified transposable elements into the classes dependingon DNA sequence homology, structural properties and mechanism of transposition. TN10 14
  15. 15. belongs to class II of transposable element with an accessory determinant of antibiotic resistancein addition to transposition functions.6.2 ObjectiveTo show the power of using the transposons to isolate mutants and to subsequently characterizethe mutants specifically in amino acid biosynthesis.6.3.1 Results and DiscussionTable 10: Number of bacterial colonies after incubation in LB and Mackonkey media 100 10-1 10-2LB Tet10 222 11 3Mackonkey 101 9 2Pale white colonies representing lactose mutants were not observed, however, white and pinkcolonies were observed in both LB and Mackonkey media respectively.Mackonkey as a selective media for enteric bacteria is supplemented with bile salts and highconcentration of sodium chloride. It is suitable for detecting lactose catabolism by noticing thecolor change from pink to red. If the lactose catabolism gene is interrupted by a transposon forexample as we anticipated, defective beta-galactosidase enzyme is produced which fermentlactose and therefore pale color cannot be observed.6.3.2 Results isolation and identification of amino acid biosynthesis mutantsTable 11: Results in MinA plates supplemented with different amino acids showingcysteine positive plates. P1 P2 P3 P4 P5P6 Cys+ Met- Ala- Lys- Gly- 1,2,3,4,5P7 His- Leu- Ile- Pro- Val-P8 Phe- Tyr- Trp- Thr- Gln-P9 Glu- Asp- Asn- Arg- Ser- 15
  16. 16. After incubating 100 colonies overnight in MinA plates supplemented with different amino acidmixtures and rich LB media replica , it was observed that some plates had colonies on LB andnot at replica spots in Min A media. Specifically, plate 1 and plate 6 had had five mutants whichcorrespond to cysteine deficient plates from the table provided. This implies that the transposoninsertion was in the cysteine biosynthetic pathway.Generalized Transduction: P1 Transduction7.1 IntroductionGeneralized transduction is a process where a bacterial DNA is transferred from one bacteriumto another using a bacteriophage, typically carrying bacteria DNA and not phage DNA. It is thepackaging of the bacterial DNA into the viral capsid and occurs either through headful packagingor via recombination.P1 phage moves genetic elements from one E.coli bacteria to another, a technique pioneered byNat Sternberg among others. P1vir is a mutant that ensures lysis of bacteria upon infection. SinceP1 packages approximately 90kb DNA, it is possible to include a selectable marker to enhancerecovery of your gene of interest. Once the phage population has been grown on donor bacteria,it is then then infected to recipient one. Through homologous recombination, the donor DNA canbe incorporated in the recipient genome. To prevent the subsequent phage from lysing theinfected recipient bacteria control of infectivity is necessary and this is achieved by regulatingthe calcium in the media since the phage requires calcium for adsorption. A calcium chelator-citrate is used to lower the amount of calcium to prevent phage adsorption but low enough tostaff the cells of calcium.7.2 ObjectiveTo use P1 to transduce an antibiotic resistance gene and confirm its location by PCR.7.3 Results and DiscussionThe following tubes were prepared and 0.1ml of each mixture cultured on LB agar supplementedwith chloramphenicol and Na-citrate. 16
  17. 17. a) 0.1 ml MG1655 + 0.2 ml of P1 vir lysate b) 0.1 ml MG1655 + 0.2 ml of P1 vir lysate c) 0.1 ml MG1655 + 100µl LB with 100mM MgSO4 and 50mM CaCl2 d) 0.2 ml of P1 vir lysates + 100µl LB with 100mM MgSO4 and 50mM CaCl2 (control)Colonies were observed in both plate A and B each containing 0.1ml of recipient E.coli MG1655strain and 0.2ml P1vir lysate from donor strain. No growth was observed in plate mixture C andD. this shows that the chloramphenicol resistance gene was transduced into the recipient E.coliMG1655 strain.Yeast agglutination8.1 IntroductionEscherichia coli have been known to agglutinate erythrocytes for a long time. In addition, theycan as well agglutinate yeast cells, Saccharomyces cerevisiae that are known to contain mannanson their cell surface and this activity can be inhibited by using D-mannose. Yeast cells has highmannose chains on their surface which make it possible for the fimbriae of bacteria to bind andcrosslink them.8.2 ObjectiveTo test the P1 transductants for their capability to agglutinate yeast cells8.3 Results and DiscussionAgglutination was not observed. However, wild type colonies formed agglutination with theyeast cells. This is attributed to the fact that they have type 1 fimbriae which bind to mannosesugar on the surface of yeast cells.The mutants on the other hand has the clustering gene interrupted by the cat cassette andtherefore cannot produce the type 1 fimbriae important for this agglutination. This explains thereason why agglutination could not be observed in the P1 transductants MG1655 with yeastcells. 17
  18. 18. Figure 5: Agglutination was not observed when the bacteria and yeast were incubated oniceReferencesAtsumi S. and JW Little (2006) Role of the lytic repressor in prophage induction of phagelambda as analyzed by a module-replacement approach. University of Arizona, USA.Gary E. Kaiser (2011) Doc Kaiser’s Microbiology, The Community College of Baltimore,Catonsville CampusHenri De Greve (2012) Virology Manual. Vrije Universiteit Brussel, Belgium.Rokney A. et all (2008) Host Responses Influence on the Induction of Lamda Prophage, HebrewUniversity, Hadassal Medical School, Jerusalem, Israel.Svenningsen SL et all (2005) On the role of Cro in Lambda prophage induction. National CancerInstitute, Bethesda, USA.Yuval E. et all (1981) Participation of Pili and Cell Wall adhesion in the Yeast Agglutinationactivity of Escherichia coli, Max-Plank Institute of Immunology, Germany. 18

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