The purpose of this lab was to find out where the mutation on a gene has occurred. This was done through the observation of mutated lac operon phenotypes and by examining various genotype growing abilities in minimal salts + glucose media, lactose MacConkey media, lactose rich media, and X-gal with minimal salts + glucose media. Four different strains labeled A, B, C, and D were used. The genotype I+P+Z+ (wildtype), I+P-Z+, I-P+Z+, and I+P+Z- were used in this experiment, and the strains had to be matched to them. The lactose MacConkey and X-gal/glucose media were used for obtaining different colored colonies. Glucose and lactose media were used to find the growth of the strains. From the comparison of Table 1 and 2, it was concluded that Strain A was wildtype, Strain B was I-P+Z+, Strain C was I+P-Z+, and Strain D was I+P+Z- strain.
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The lac operon is part of a gene that codes for proteins that are required for lactose digestion and regulation of that operon. The absence of this operon would result in bacterial mutations. The bacteria would not be able to metabolize lactose and would have to find other means of obtaining energy when glucose is not present. The lac operon consists of the lac Z, Y and A gene. The lac Z gene codes for beta-glycosidase that hydrolyze the disaccharides. The lac Y gene codes for permeases that is used to raise the permeability of beta-galactosidase into the cells. The lac A gene is the final region, which codes for transacetylase. The Z and Y genes are not often expressed because the repressor protein (I) is bonded to the place where transcription starts, the promoter region (P). In the presence of lactose, the repressor protein (I) binds and starts transcription.
The aim of this experiment was to find out where the mutation on a gene has occurred. This was done through examination of mutated lac operon phenotypes as well as examination of various genotype growing abilities in minimal salts + glucose media, lactose MacConkey media, lactose rich media, and X-gal with minimal salts + glucose media. If the mutation had occurred in the repressor protein (I) gene, the repressor protein would not bind to the promoter and continual transcription would occur. If the mutation had occurred in the promoter region (P) in lac Z gene, the repressor protein would be unable to bind to the operator and RNA polymerase would have trouble binding to the promoter region. If the mutation had occurred in structural gene (Z), the beta-galactosidase would not be formed; therefore lactose would not be fermented.
Materials and Methods:
In order to perform this experiment, four media plates including minimal salts + glucose media, lactose MacConkey media, lactose rich media, and X-gal with minimal salts + glucose media were obtained. These were used to identify the genotypes of the four different bacterial strains. The four strains had I+P+Z+ (wildtype), I+P-Z+, I-P+Z+, and I+P+Z- genotypes. The + indicated that the bacterial strain was normal for that gene, while the ââ‚¬" indicated that the bacterial strain contained mutation in that gene. The strains were labeled with A, B, C, and D, but not with its genotypes. Each of the four strains were streaked on each of the four media plates. The 16 plates were then incubated at 37ËÅ¡C for one to two days and analyzed. The minimal salts/glucose and the lactose rich media plates were checked for the growth, while the lactose MacConkey and X-gal with minimal salts/ glucose media plates were checked for color.
Table 1: Predictions for Each Strand in Each Medium
Will it grown on Minimal/Glucose?
What color will the colonies be on MacConkey
What color on Lactose MacConkey?
What color on X-gal/Glucose?
White/light blue Colonies
White with pink
Weak Red small Colonies
Heavy Red Colonies
Heavy Blue Colonies
Always on Time
Marked to Standard
White Colonies & Orange Media
Caption: Predictions were made from the protocol and the help of the lab TA.
According to Table 1, the wildtype strain (I+P+Z+) can grow on glucose as well as lactose media plates and has red and blue color on lactose MacConkey and X-gal/glucose media plates. I+P-Z+, I-P+Z+, and I+P+Z- can grow in glucose media plates. Except for I+P+Z-, all of the other strain can grow on lactose media plates. I+P-Z+, I-P+Z+, and I+P+Z- have weak red, heavy red and white color colonies in the lactose MacConkey media plates. The I+P+Z- have changed the color of the media from red to orange. I+P-Z+, I-P+Z+, and I+P+Z- have weak blue, heavy blue and white color colonies in X-gal/glucose media plates (Table 1). Our result was similar to that expected and stated above.
Table 2: Phenotypic Observation of Plates
Lactose Rich/ MacConkey
Caption: The strains were streaked on four different plates and incubated at 37ËÅ¡C for one to two days. The observations were recorded after the incubation period.
According to Table 2, strains A, B, C, and D all grew in minimal/glucose media plates. Except for Strain D, all of them grew in lactose rich media plates. Strain A, B, C, and D had red, heavy red, weak red and white color colonies in lactose MacConkey media plates. Strain D had changed color from red to orange. Strains A, B, C, and D had white, heavy blue, weak blue and white color colonies in X-gal/glucose media plates. (Table 2)
Table 3: Identifications of the Strains
Mutated Repressor Protein gene
Mutated Promoter P of Z gene
Mutated Structural gene of Z gene
Caption: The identification of the strains was made from data in Table 1 and 2.
According to Table 3, we can conclude that Strain A was the wildtype, Strain B was the I-P+Z+, Strain C was the I+P-Z+, and Strain D was I+P+Z-. (Table 3)
Figure 1:- Strain B I-P+Z+
Caption: - Picture result of Strain B
Figure 2 :- Wild type, I+P+Z+
Caption :- Picture Result of Strain A, Wild type.
Figure 3:- Strain D, I+P+Z-
Caption :- picture result of Strain D, I+P+Z-
Figure 4: - Strain C, I+P-Z+
Caption :- Picture Result of strain C
Conclusion was reached for all the strains and their identification with the help of data obtained in Table 2 and 3. All the strains were different because the media had impacted their phenotypes. Bacteria donââ‚¬â„¢t use lac operon if glucose is present and lactose is absent; hence all the strains grew on the minimal salts/glucose media plates. Strain D did not grow on the lactose rich media because of the mutation in the structural genes of lac Z gene. The repressor protein (I) and the promoter (P) were functioning well, but with mutation in structural genes (Z), the RNA polymerase was unable to make the proper mRNA, which could not make the beta-galactosidase. Therefore, bacteria in Strain D were unable to ferment lactose resulting in no growth. (Table 2)
In the lactose MacConkey media, the bacterial colonies turn red when they make beta-galactosidase because there is low pH. When beta-galactosidase digests lactose, some acid is produced that can lower the pH in the media. Strains A, B, and C colonies turned red, because they all produce beta-galactosidase to some extent. Strain A was the wildtype strain with properly functioning lac operon, having normal red colonies. Strain B had a mutated repressor protein (I), which led to extremely high amounts of beta-galactosidase being produced, which therefore made the colonies from that strain appear heavy red. Strain C had a mutated promoter (P), which made few beta-galactosidase because the repressor protein and the RNA polymerase had difficulty biding to the operator and promoter. Due to the low amounts of beta-galactosidase made, a reduced amount of lactose was fermented and the pH was not lowered to the extremes, which made the colonies appear weak red. At last, Strain D had white colonies. Bacteria in Strain D had to use peptone to grow because of the low amount of lactose fermentation due to the reduced amounts of beta-galactosidase. The fermentation of peptone raises the pH, which makes the MacConkey media turn orange, and the colonies turn red. (Table 2)
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The X-gal/glucose media plate had glucose as well X-gal, which is a precursor of lactose. The bacteria preferred to use glucose over X-gal. Bacteria, which have the ability to make beta-galactosidase, can ferment X-gal and become blue in this media. Strain A was the wildtype strain, so with the presence of glucose did not use the lac operon. Strain B had mutated repressor protein (I) and hence continually produced beta-galactosidate, which made excessive beta-galactosidase that fermented high levels of X-gal making its colonies show heavy blue color. Strain C had mutated promoter (P), which made few beta-galactosidase that fermented minimal X-gal making and made its colonies show light blue color. At last, Strain D with mutated structural gene (Z) had no beta-galactosidase, and it fermented only glucose, hence made its colonies appear white. (Table 2)
The data from Table 2 was compared to the data from Table 1, from which it was concluded that Strain A was the wildtype, Strain B was the mutated repressor protein strain (I-P+Z+), Strain C was the mutated promoter strain (I+P-Z+), and Strain D was the mutated structural gene strain (I+P+Z-). (Table 3)