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Raunvísindadeild; Líffræðiskor Erfðafræði Verkleg Erfðafræði Conjugation in E. coli Námsbraut: Raunvísindadeild; Líffræðiskor Námskeið: 09.51.35 Erfðafræði Nafn kennara: Ólafur S. Andrésson, Zophonías O. Jónsson, Sigríður H. Þorbjarnardóttir og Bryndís K. Gísladóttir. Vikudagur, hópur: Fimmtudagur, síðari kennslustund, Hópur 2 og 4. Tilraun framkvæmd: 18., 25. september, og 2. október, 2003 Skýrsluskil til kennara: 16. október, 2003 Skýrsluskil til stúdents: Einkunn: Nöfn stúdenta: Bjarki Steinn Traustason, Egill Guðmundsson, J. Gabriel-Rios Kristjánsson, Marcella Manerba og Nicoletta Palmegiani. ________________________________ Bjarki Steinn Traustason ________________________________ J. Gabriel-Rios Kristjánsson ________________________________ Egill Guðmundsson ________________________________ Marcella Manerba ________________________________ Nicoletta Palmegiani Háskóli Íslands Raunvísindadeild; Líffræðiskor 4 Erfðafræði Conjugation in E. coli Introduction: E. coli’s chromosome is one continuous DNA-molecule, about 1.3 mm on length. In the cytoplasm of some E. coli-strains, is a so-called F-factor which is a small circular DNA molecule which goes under replication independent to the chromosome’s replication. The Bacteria which have the F-factor are called F+, but the ones without it are called F–. The F-factor encourages to conjugation between F+- and F–bacteria, when they are cultured together. At conjugation, the F-factor transfers into the F–-bacterium and it will become F+. The (first) F+-bacterium retains at least one copy of the F-factor and remains F+. Once in a while (in about, every (other) 105 cell) recombination occurs between the F-factor and the bacterium’s chromosome. During conjugation, a part of the chromosome can transfer to the F–-cell along with the F-factor. In Hfr-strains, the F-factor has integrated into the bacterium’s chromosome and persisted there for generation after generation. In these strains, each and every cell can transfer it’s genetic material if it reaches conjugation with F–-cell. Hfr-cell traslocates it’s genes in certain order at the same time the chromosome is replicated. Replication and translocation starts in certain ‘set-point’ in the integrated F-factor and can continue until the whole chromosome has been transferred, and the F-factor amongst it. The translocation of the whole chromosome takes about 100 min at 37 °C, but the connection (in conjugation) between Hfr- and F– -cell frequently rupture/uncouple before the translocation for the whole chromosome has concluded. After the genes from F+- or Hfr-cell have been transferred into F–-cell, they can recombinate into the F–-chromosome. The gene which have been incorporated, inherit to the next generation but the others are destroyed. In this exercise, we monitor gene translocation from E. coli-Hfr cells (CGSC6026) to E. coli-F– cells (CGSC1157). The F–-strain is auxotroph, concerning the amino acids, threonine, leucine, proline, and histidine, but it is, however, resistant to streptomycin, because it has the rpsL mutation in ‘ribosome protein’-gene. The Hfr-strain is streptomycin-sensitive, which is a dominant feature. By incubating on various medium, you can see a gradient figure in the number of colonies which grow, reckon with the genes’ loci in the chromosome, which was transferred to the recipient. Abbreviations: E. coli, Escherichia coli; F-factor, fertility factor; Gal, galactose; His, histidine; Hfr, high frequency of recombination; Leu, leucine; Pro, proline; Thr, threonine. Aims/hypothesis: The aim, is to determine bacterial conjugation, the order of gene translocation from Hfr-E. coli strains to F–-strains, and to localise genes on E. coli’s chromosome. Design and Methods: Reference to work sheets in manual booklet, for present exrecise (p.18-20). Exception from the 6th part, p.20, where petri dishes were incubated with 0.1 mL of every dilution. Used dilutions were: for pro–-dishes, incubated separately with 10–3 and 10–4 dilutions; instead of 10–2 and 10–3 dilutions. Using more dilute solutions makes the counting of colonies easier, which leads up to more intuitive viable count. Háskóli Íslands Raunvísindadeild; Líffræðiskor Erfðafræði Results: TABLE 01, E. COLI’s VIABLE COUNT IN DIFFERENT MEDIUM: pro+ type: colonies dilution: Group: H1 H2 H3 H4 H5 (thr/leu)+ his+ colonies colonies 10–3 /dish 10–4 viable c. 10–2 /dish 10–3 viable c. 10–1 /dish 10–2 viable c. 173 192 220 186 296 10 17 29 34 31 1.7106 1.9106 2.2106 1.9106 3.0106 828 - 273 192 205 280 362 2.8106 1.9106 2.1106 2.8106 3.6106 11 281 126 9 284 1 5 0 0 10 1.1103 5.0103 1.3103 9.0102 1.0104 2.1106 2.6106 3.7103 Viable count, general formula: (colonies ∙ (volume of solution / volume used for incubation)) / dilution. Eg,: viable count = (192 colonies ∙ (1.0 µL solution / 0.1 µL used for incubation)) / 10–3 = 1.9 ∙ 106. <viable count>: TABLE 02, CONTROLES, NUMBER OF COLONIES PER PETRI DISH: type: strain: 6026 1157 pro+ (thr/leu)+ his+ 0 0 0 1 0 0 TABLE 03, PARALELLISM OF COLONIES IN DIFFERENT MEDIUM, AFTER REPLICA PLATING: Group: pro+ gal+ his+ pro+ gal+ his– pro+ gal– his– pro– gal+ his+ pro– gal– his+ pro– gal+ his– pro– gal– his– Σ H1 H2 H3 H4 H5 Σ 1 3 24 1 0 2 19 50 2 8 17 0 0 0 23 50 1 4 19 1 0 0 25 50 1 7 21 1 1 0 19 50 2 7 22 0 0 2 17 50 7 29 103 3 1 4 103 250 TABLE 04, NUMBER OF COLONIES PER PETRI DISH, WHICH GROW AFTER REPLICA PLATING: type: Group: H1 H2 H3 H4 H5 Σ pro+ gal+ his+ 28 27 24 29 31 139 7 10 6 9 11 43 2 2 2 3 2 11 TABLE 05, THE RATIO FOR THE TYPES OF COLONIES: Total, from H2 type: pro+ : gal+ : his+ : ( 27/250) ∙ 100% = 54.0% ( 10/250) ∙ 100% = 20.0% ( 2/250) ∙ 100% = 4.0% Total, from H4 type: pro+ : gal+ : his+ : ( 29/250) ∙ 100% = 58.0% ( ( 9/250) ∙ 100% = 18.0% 3/250) ∙ 100% = 6.0% ... in comparison Total, from all groups type: pro+ : (139/250) ∙ 100% = 55.6% gal+ : ( 43/250) ∙ 100% = 17.2% his+ : ( 11/250) ∙ 100% = 4.4% general formula: (the total number of specific type of colonies / the total number of all types of colonies) ∙ 100% Háskóli Íslands Raunvísindadeild; Líffræðiskor Erfðafræði FIGURE 01, DETERMINATION OF THE LOCATION OF THE proA-GENE AND THE galK-GENE, COMPARED WITH THE EQUATION OF THE COLONY-FUNCTION: Determ. of proA: From Table 04, y = 139 (ie, the total number of colonies, type pro+). The x-value is the unknown, and locates the minutes. Determination of proA number of colonies 1000 y = 209.93e-0,0703x 100 10 1 0 10 20 30 40 50 t [min] Determ. of galK: From Table 04, y = 43 (ie, the total number of colonies, type pro+). The x-value is the unknown, and locates the minutes. Determination of galK number of colonies 1000 y = 240.25e-0,0704x 100 10 1 0 10 20 30 40 t [min] y = 209.93 · e–0.0703x ; y/209.93 = e–0.0703x ; ln(y/209.93) = –0.0703x ; x = ln(y/209.93) / –0.0703 ; x = ln(139/209.93) / –0.0703 x = 5.8649 x~6 50 y = 240.25 · e–0.0704x ; y/240.25 = e–0.0704x ; ln(y/240.25) = –0.0704x ; x = ln(y/240.25) / –0.0704 ; x = ln(43/240.25) / –0.0704 x = 24.439 x ~ 24 Conclusion/Discussion: The colonies which grow on the agars, are only the F–-strains, through conjugation have gotten their genes, which allow the biosynthesis of the amino acids: threonine, leucine, proline, histidine. Hfr-strains don’t grow in the agar because it contains streptomycin, which the bacteria are sensitive to. The control dishes show that the strains are correctly defined, ie, the Hfrbacteria are streptomycin sensitive and the F–-strain is auxotroph that needs the amino acids above-mentioned to grow. Because of the Hfr-strain is streptomycin sensitive we shouldn’t expect any colonies to grow on the dishes. From Table 02 ■, evidently one colony grew in one of the control dishes, which is plausibly contamination, judging from it appearance. Estimating from our numbers of colonies that can grow on different agars, and our given temporal order of inheritance, we can guestimate were the F-factor was incorporated into the recipient’s chromosome. Based on the higest average data of viable count, Table 01 ■, we can assume that the F-factor was integrated upstream to the thr/leu-genes. In fact those genes adjacent to O (origin to translation) are transferred first, leading the mobilized chromosome into the F–-bacteria. The F-factor therefore becomes the end of the broken chromosome, and is the last part to be transferred. This proposal explains why the recipient cell, when conjugated with Hfr, remains F–. The second highest mean of viable count came from the colonies which grew on the pro–-agar, and lowest mean of viable count was on the his–-agar. Ie, the Háskóli Íslands Raunvísindadeild; Líffræðiskor Erfðafræði assumed order is: thr/leu → pro → his. The Ratio given in Table 05 ■, also confirms the order, in addition to, applying galK into the order. The translocation of the genome starts at 98th min in Hfr-strains and it is likely that the F-factor has integrated near that location. Answers to Questions: 1. In what part of the chromosome is the F-factor integrated, in Hfr-strain CGSC6026? The F-factor has integrated near that location at 98th min. 2. Check the results from the petri dishes, which were incubated after conjugation. What is the appearent order of gene translocation? The assumed order is thr/leu → pro → his. 3. Observe data resulting from using replica plating-method. What is the most likely order of the genes? Is the order the same as data from incubation after conjugation dictate? Our results confirm the given order of the genes. 4. Why aren’t all pro+-blots also (thr/leu)+? Sometimes recombination occurs in the chromosome between thr-gene and leu-gene so they do not come through, which means that some colonies can be pro+ and (thr/leu)– at the same time. 5. What is the function of the controls? The control dishes show that the strains are correctly defined. 6. If the location of leuB, his, and galK is given, on what minute does proA localise, according to frequency-data from replica plating? According to graphic calculations, proA is on 5.86th min, ~ 6th min. Figure 01 ■. 7. How did the mapping of galK come along? According to graphic calculations, galK is on 24.4th min, which is far from the right value, 17th min, Figure 01 ■. When we calculated the position of the hisgen we got a value which was also far from the right value. This probably due to the long distance from the origin of the translation. The error is also evident by comparison of the line-equations, focusing on different slope’s values. 8. How do your results conform with what is known, by contrast to, eg, information on p.17 and 18 in manual booklet? The regularisation is good. □ ■ Háskóli Íslands