2. You have isolated a new organism (B. sanfranciscus) from soil. You find that

Question

2. You have isolated a new organism (B. sanfranciscus) from soil. You find that it can be grown in the lab on plates of LB media and it also grows well on M9 media (see Table 4.2 in the text). You decide to identify the genes in the organism that are necessary for the biosynthesis of the amino acid arginine using a series of steps similar to that diagrammed in Fig 12.1 of the text. You start by infecting B. sanfranciscus with a defective phage carrying the transposon Tn10 Briefly describe how you will select for B. sanfranciscus clones that have had Tn10 transposition into their chromosomes. What media will you use? A. Problem Set 4, Spring 2018 B. Briefly describe how you will screen for B. sanfranciscus clones that have had the genes involved in arginine biosynthesis tagged with Tn10 C. After a lot of work, you identify 7 clones that are unable to synthesize arginine. You analyze all seven clones by 2 dimensional gel electrophoresis (2D gels; see Fig A3.4). For six of the clones, each clone can be correlated with a single protein on the 2D gel - ie. -a spot that is present in the wildtype strain is missing in the Tn10-mutagenized clone; in the seventh clone all six proteins are missing. What does this tell you about the organization of the genes encoding the proteins responsible for arginine biosynthesis in B. sanfranciscus?

Explanation / Answer

2) A)- a quick method for screening would be by colony PCR using Tn10 specific primers. The bacteria will be grown on LB media, as they may lack mutated for biosynthetic genes necessary to support growth.

B) For this, bacteria will be grown on M9 medium supplemented with all the amino acids. The growing colonies will then be replica plated onto M9 medium lacking arginine. The colonies that are not able to grow will be selected from master plate. These will be verified by growth on medium lacking other amino acids but arginine. The selected mutants will be in arginine biosynthesis pathway.

C) The information deduced:

1) Arginine biosynthesis in this particular species may involve six steps, hence six different proteins giving similar phenotype.

2) Since the seventh mutant lacked all the six proteins, it means they are arranged in an operon that has a common regulator. The seventh clone may have a cis mutation in the promoter or operator sequence of the operon. The seventh mutant could, also, be in an activator protein needed to activate the operon.

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