Question

1. Currently you are studying threonine synthesis in E. coli. To find genes whose protein products are important for threonine synthesis, you perform a mutagenesis to find mutants that require a source of threonine to grow.

(a) Immediately after mutagenizing the bacteria, would you choose to grow the bacteria on rich media or on minimal media? Briefly explain your choice.

Rich media. A mutant unable to make a particular amino acid will not be able to grown on media lacking that amino acid (minimal media). Therefore, to isolate such mutants, they would first have to be grown on media containing the amino acid they are unable to make themselves. To identify the mutant phenotype, you would then have to find colonies that grow on rich media but NOT on minimal media. You can do this using a technique called ‘replica plating.’ This technique involves moving mutatagenized bacteria plated on rich media by ‘printing’ the colonies onto a new plate of minimal media (using a fine velvet ‘stamp’).

You find a total of nine mutants, all of which have recessive phenotypes. You perform complementation tests with all of your mutants.

(b) What information do the complementation tests provide?

Provides information about how many genes or functions might be involved in a specific process. When mutants are part of the same complementation group (in other words: fail to complement one another), we can infer they have mutations in the same gene. So, this allows us to get a minimum estimate of the number of genes involved in our pathway of interest.

Below are the results for your complementation tests. A (+) means growth and a (-) means no growth:

	mt1	mt2	mt3	mt4	mt5	mt6	mt7	mt8	mt9	wt
mt1	-
mt2	+	-
mt3	-	+	-
mt4	+	+	+	-
mt5	-	+	-	+	-
mt6	-	+	-	+	-	-
mt7	+	+	+	+	+	+	-
mt8	+	-	+	+	+	+	+	-
mt9	-	+	-	+	-	-	+	+	-
Wt	+	+	+	+	+	+	+	+	+	+

Question 1 continued

(c) How many complementation groups did you identify?

4. We arrive at this number by looking at each pair of mutants and looking for complementation between them (growth phenotype). A mutant will never complement itself, hence all ‘-‘ (no growth) along the diagonal. Proceeding down the rows of the table, we first see that the combination of mt1 and mt2 is able to grow. Therefore mt1 and mt2 complement each other, and are defined as being in different complementation groups (in other words: different genes). In the next row, we see that mt3 and mt1 fail to complement, therefore they are in the same complementation group (in other words: in the same gene). However, mt3 and mt2 do complement each other, so they are in different complementation groups, which we could have guessed from what we concluded from the previous row. Continuing down the table like this:

Question #1   How many complementation groups are there?

Question #2   For each complementation group, indicate which mutants belong to that complementation group.

Group 1 - mt4

Group 2 - ________________________

Group 3 - ________________________

Group 4 - ________________________

Question #3 How many gene involved in the synthesis of threonine have you identified?

Answer 1