Lac operon and metabolism of lactose in different environments

PROBLEM SET

1 Experimental Background

E. coli produces beta-galactosidase when lactose is present. X-gal is a colorless, lactose-like
molecule that can be split into two fragments by beta-galactosidase. One of these product
molecules is blue. We identified two genes, A and B, that might be involved in the lactose
processing in E. coli. Using CRISPR/Cas9 techniques, we sufficiently produced different cell
lines that had double-stranded breaks (DSBs) in genes A and B. After recovery, we plated the
modified colonies on agar, containing glucose and corresponding treatment.

Table 1. Results. White and blue demonstrate the quantification of colored colonies.

Treatment	Control		Gene A DSB		Gene B DSB		Gene A/B DSB
	White	Blue	White	Blue	White	Blue	White	Blue
No Lactose	+++	– –	+++	—	+++	—	+++	—
Lactose	+++	– –	+++	—	+++	—	+++	—
No Lactose + X-gal	+++	– –	+++	—	+++	+	+++	—
Lactose + X-gal	+++	+	+++	—	+++	+	+++	—

Table 2. List of possible genes by mass spec.

Lac-A	Thiogalactocide transcetylase
Lac-I	Regulator gene
Lac-O	Operator gene
Lac-P	Promoter gene
Lac-Y	Lactose permease
Lac-Z	Beta-galactosidase

Furthermore, studies have found out that there might be a competing interaction between the
usage of glucose and galactose. We identified an important gene in this regulatory pathway, and
we performed a DSB within this gene.

Table 2. Results. Blue colonies on agar plates were counted and quantified under the Control and Gene C DSB column.

	cAMP level	Control	Gene C DSB
Glucose +/ Lactose –	—	—	—
Glucose -/ Lactose +	+++	+++	—
Glucose +/ Lactose +	—	+	—

Figure 1. Experimental Setup.

2 Problem Set

Question 1: Based on the Table 1 and Figure 1 provided, speculate the functions of gene A and B and give the name of the gene. If possible, draw the corresponding construct, i.e., the gene and the location of the DSB.

Question 2: Why are there fewer blue colonies in the Glucose+/Lactose+ group than in the Glucose-/Lactose+ group?

Question 3: Based on the different results in Experiment One and Experiment Two, what is the fundamental difference between Gene A/B and Gene C? (Hint, think from the gene regulation, rather than the outcomes)

Solution

Gene A: Lac-Z, Beta-galactosidase
Gene B: Lac-I, regulatory control of the expression of Lac-Z

Glucose is preferred over lactose. The agar plates contains X-gal, which will turn blue if
bacteria are actively transcribing beta-galactosidase. Thus, more blue colonies means more
lactose usage. Glucose+/Lactose+ shows fewer blue colonies, meaning that less lactose is
used in the presence of glucose. One might speculate that the reason behind this is that
bacteria use glucose more readily. Lactose needs to be converted to glucose and galactose
before it can be used by bacteria.