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sources: here, here and here.
The destinies mend rifts in time as men etches fate anew
(a) Transposon method: Assume the original host is resistant to chloramphenicol. A suicide plasmid carrying a transposon would be transferred cells of the host bacterium by transformation or conjugation from an E. coli culture. Let us assume that the transposon also carried a tetracycline resistance gene. The transformed celles (or the tranconjugants) would then be plated on growth medium containing tetracycline. For the tetracycline gene to survive, the transposon must hop out of the suicide plasmid into othe host DNA. Since this random event will occur in many millions of cells, some cells are likely to contain transposon insertions in the putative Cm gene. We can select for cells containing insertions in the Cm gene by plating all tetracycline resistant cultures on duplicate plates containing both tetracycline plus chloramphenicol. Cultures that do not grow in the presence of Chloramphenicol must contain insertions in DNA involved with Cm resistance. To confirm that the transposon had inserted into the Cm gene, we could use PCR technology to sequence DNA flanking the transposon to determine whether it is identical to that obtained previously (see question).
(b) Single crossover (or cointegrate method). Assume the original host is resistant to chloramphenicol. A suicide plasmid containing part of the cloned gene could be introduced into the original host by transformation or conjugation. Assume the plasmid also has a tetracycline resistance gene for selection purposes. When the transconjugants or transformants are grown on medium containing tetracycline, the only way the plasmid DNA can survive is by integrating into the host DNA. This will be facilitated by recombination between the part copy of the Cm gene and the wild type copy in the host. This event will result in integration of the entire plasmid into the host DNA, thereby creating an insertional mutation. Tetracycline resistant cultures would then be plated on medium containing tetracycline + chloramphenicol. Lack of growth would confirm that the Cm gene had been insertionally inactivated. This could be confirmed by PCR amplification of DNA encompassing the point of insertion of the plasmid DNA followed by sequencing of the host DNA flanking the insertion. If the sequence data was identical to that obtained previously (see question) then you could say that the gene encoded Chloramphenicol resistance.
(c) Allelic replacement. Assume the original host is resistant to chloramphenicol. The cloned copy of the gene would first be insertionally inactivated in E. coli by cloning a copy of another gene eg tetracycline resistance gene into the Cm gene. The construct would then be cloned into a suicide plasmid (carrying an ampicillin resistance gene for selection of the plasmid) and transferred to the host cells by conjugation or transformation. Transformants or transconjugants would be selected by plating on tetracycline medium. Tetracycline resistant colonies would then be plated separately on growth media containing tetracycline + chloramphenicol and Tetracycline + ampicillin. If a double crossover recombination event had resulted in exchange of the mutated copy of the Cm gene into the host, then no growth should be observed. This would be evidence that the Cm gene encoded chloramphenicol resistance. This could be confirmed by sequencing across the junction between the tetracycline gene and flanking DNA. Any cultures that were able to grow on Tet + ampicillin would represent cointegrates formed by single crossover recombination events. These should also be chloramphencicol sensitive.
MYM|Fear (Top)
MYM|LevenT (Top)
MYM|MaNia- (Middle)
MYM|H4nn1- (Roam)
MYM|Maelk (Bottom)
.........................................................................inF.SniFFeR- (Top)
inF.CAPSLOCK (Top)
inF.keRm (Middle)
inF.JumpEr (Bottom)
Arte (Bottom)
Source: ANN
Number of episodes: 13
Spice and Wolf is by far one of the best shows I’ve watched in 2007/2008. One thing I like about this series is the theme. It’s quite rare to see an anime using economy, merchant and trading as a theme, yet manage to give fairly good impression toward the audience. This theme may be encompassed in a possibly generic storyline - a young merchant named Lawrence travels across the cities to trade items and later meet a wolf-deity girl named Horo, and together they start the journey to the north, the place where Horo belong; however what makes it interesting is the series of events happened along their journey from town to town. It’s all about profit!
Story: This area gives me a mixed bag of feeling, but overall I’m more on the positive side. It’s always a pleasure watching the banter of Lawrence and Horo in almost every episode, having some nice conversations and critical analysis on the economic stuff then ran into. Horo is undoubtedly the center of attraction in almost every episode, showing a strong characteristic and pride (that is, in her tail). Her loneliness which was depicted in several occasion were outweighed by her bright, adorable and cheerful characteristic shown whenever she spend her time with Lawrence. The voice Actor (Ami Koshimizu) did a splendid job to give Horo an outstanding characteristic. It appears that Horo voice sounds more erotic and appealing that what the subs appear, so it’ll be more enjoyable if I fully understand what the character says. As for the economic stuff, I wish they could give a better and creative means to convey them to the audience, like what Hikaru no Go did with Go board game. Sometimes the way they tell the details about the trade, the silver content and such are very linear and boring. Well at least to some extent Horo did a good job to explain certain thing in a simpler way to solve the puzzles. And lastly, it seems to me that due to the short length of this anime, sometimes the story looks compressed that some details are failed to be covered. Cloe for example, I think she plays very little role in the overall story of Spice&Wolf. She appears early in the series as
While the animation quality is not something to be proud of particularly in character animation area, the way they describe the Medieval Europe setting is very impressive, with in depth detail of building structures and environment. Character design is fairly simple, reminding me a lot to Princess Resurrection. The main characters (Horo, Lawrence, Nora and Cloe) somewhat gain the most out of it, making most of the side characters a bit disappointment with many of them lacks variation and look unappealing.
As for the music selection, I have to say that the BGM used suits very well with the scenarios and theme of the series, especially because they’re using actual instrument rather than synthesizer. While many of the tracks are not really fit to be put on your music playlist, they certainly sounds unique to Spice&Wolf and likely more recognizable, just like the unique BGM tracks in titles like Rozen Maiden, Mai Hime and Hayate no Gotoku. The opening song, Tabii no Tachuu by Kiyoura Natsumi is one of the sweetest OP I’ve listened to, and while I’m not that fond with the ending song (Ringo Biyori by Rocky Chack due to the “Engrish-ness” thus made me hard to follow the song without actually reading the lyric. But somehow I grew attached to the song after listening to the full version of the song, although if you read carefully to the lyric, it kind of does not make much sense to the story.
Overall Spice and Wolf is very enjoyable show to watch. Sure it would be boring if you didn’t enjoy the economic stuff, but for me the presence of Horo is enough to make this show interesting in its own way. In my anime dictionary, I would normally give this one a 10 out of 10 rating, but considering some weaknesses that this series posses, I would give this 8.5/10. I hope with that kind of ending shown in the final episode, it will open an option for second season, which I would be more than happy if it really comes out.
Spice&Wolf OP: Tabi no Tachuu