The Gene Control Process in Bacteria - Term Paper Example

Lab Report: Gene Control in Escherichia coli- Lac Operon

Introduction

The gene control process in bacteria is an unusual activity that often deviates from the typical protocols witnessed in multicellular organisms. Bacteria, like E. coli, often lack the ability to manufacture all the necessary proteins all times. However, they adapt to their surroundings by controlling particular proteins in certain environments. These proteins that are assembled only when needed are often the end-products of the inducible genes. Good examples of inducible genes include the bacterial enzymes necessary for the metabolism of particular sugars as a source of energy. This process is termed as a negative gene control system because it involves inducer-repressor control mechanisms. In fact, gene expression is in most cases blocked. On the other hand, proteins synthesized under all conditions fall under the category of constitutive genes, such as the enzymes needed during glycolysis. These systems in prokaryotes are categorized under the positive gene control mechanisms, mainly because they do not require activators and terminators (Ellison et al., 1997).

One of the best examples of protein synthesis that involves inducible genes includes the manufacture of β-galactosidase, an induced enzyme, in the lactose system of E. coli. The process is currently used as a model system for studies involving mechanisms of gene control in bacteria and other prokaryotes (Griffiths, 1996). The entire lactose metabolism system is controlled by a lac operon. An operon is principally a group of genes that generate proteins with related functions or roles (Ramey, 2002). Primarily, the lac operon consists of three structural genes that code for proteins used in lactose metabolism. The first one is the lacZ gene that codes for β-galactosidase, a protein that hydrolyses lactose into galactose and glucose. The second structural gene is the lacY that codes for a β-galactoside permease, an enzyme critical in the uptake of lactose. The last structural gene is lacA that codes for a transacetylase. Additionally, it is important to note that a lac operon must have an inducer, a terminator, a regulator, as well as an operator (Saier, 1996).

Therefore, the primary purpose of this experiment is to investigate the process of negative gene control in E. coli. In this particular study, Isopropylthiogalactoside (IPGT) is principally used to induce the synthesis of β-galactosidase. Thus, it is expected that the levels of IPGT reduce as the process continues, especially as more and more lactose is converted into glucose and galactose. On the other hand, chloramphenicol is used to inhibit the process of protein synthesis in E. coli. Finally, sodium azide is critical in blocking the final step of the ATP synthesis, particularly the last step in the electron transport chain.

Methodology

• Preparation of E. coli Sample Cultures
• Materials required
• Five flasks containing 20ml of E.coli culture which has been grown at 37° C
• 0.1% solution of cetyl trimethyl ammonium bromide
• IPTG Solution
• IPTG Solution containing sodium azide
• IPTG Solution containing glucose
• Chloramphenicol
• Procedure

Five labeled test tubes, each containing 60ul of CETAB, were set ready to collect samples from the five cultures. The following were added to the cultures:

CULTURE

ADDITION

Control

5.0ml water

IPTG {1}

5.0ml IPTG solution

IPTG{cm}

5.0ml IPTG solution, chloramphenicol added later

IPTG + Azide

5.0ml IPTG solution containing sodium azide

IPTG + glucose

5.0ml IPTG solution containing glucose

The contents were then immediately mixed after which 1.0ml of the sample was added to each culture using an automatic pipette. The time of each addition was noted. The flasks were afterward placed in a shaking water bath and incubated. After mixing the cultures, 1.0ml samples from each container were taken at 15 minutes intervals for one hour. The samples were mixed with CETAB and stored on ice. After 35 minutes, five drops of concentrated chloramphenicol solution {conc Cm} were added to the IPTG culture only, and the sample returned to the water bath ("Beta-Galactosidase Activity Assay," 2016).

• Measurement of β-galactosidase using ONPG
• Materials
  • • • Samples of E.coli on ice
      • O-nitrophenyl B-galactoside {ONPG} SOLUTION
      • 1M sodium carbonate
• Procedure

The tubes containing E.coli were placed in a water bath at 37c and left for 5 minutes to bring the contents to the working temperature of the assay. Afterward, 0.5ml of ONPG was added at 30 seconds interval to each tube and the contents mixed thoroughly. The tubes were then incubated for 15 minutes at room temperature. The reaction was stopped eventually, by the addition of 0.5ml of 1M sodium carbonate at 30 seconds intervals and the contents mixed ("Beta-Galactosidase Activity Assay," 2016). The spectrophotometer was set to read absorbance at 420nm. Time sample from each flask was used as the blank to zero the spectrophotometer. The absorbances of the incubated samples were read separately and recorded.

Results

The following results were obtained for β-galactosidase activity which was shown by its absorbance at 420nm after been incubated with ortho-nitrophenyl B-galactoside.

Time {minutes

Water+ E. coli

IPTG+E. coli

IPTG + E. coli + Cm at35 mins

E. coli + Azide + IPTG

glucose +E. coli + IPTG

0

0.000

0.00

0.00

0.00

0.00

15

0.000

0.072

0.116

0.005

0.005

30

0.000

0.020

0.217

0.00

0.009

45

0.000

0.285

0.257

0.018

0.009

60

0.000

0.385

0.274

0.015

0.013

Description

Treatment

Description

Treatment One (Water+ E. coli)

No activity at all.

Treatment Two (IPTG+E. coli)

A steady increase in the yellow colour with time demonstrated by the increasing absorbance.

Treatment Three (IPTG + E. coli + Cm at35 mins)

A steady increase in absorbance until the 35th minute.

Treatment Four (E. coli + Azide + IPTG)

Little activity.

Treatment Five (glucose +E. coli + IPTG)

Little activity.

Discussion

From the experiment, it is evident that the extent of the activity (measured by the amount of yellow color and absorbance) is directly proportional to the presence of the inducer (Isopropylthiogalactoside). This study is, therefore, a typical example of a lac operon. Thus, the amount of IPGT (β-galactoside) present reflects the production of the β-galactosidase enzyme necessary in the hydrolysis of lactose into galactose and glucose. Lack or inhibition of the inducer means the absence of the β-galactosidase enzyme hence a reduced production of the yellow product, o-nitrophenol (ONP).

For example, in the treatment involving water and E. coli only, there is significantly no activity observed because there is no inducer (IPGT) to trigger the production of β-galactosidase that is responsible for the hydrolysis process. Therefore, absorbance at all times remains at zero (see appendix one). This particular treatment is primarily used as a control experiment. Similarly, in the third treatment (involving E. coli, IPGT, chloramphenicol), the absorbance is steady until the 35th minute when chloramphenicol is added. The primary reason there is less activity between the 35th and the 60th minute is that chloramphenicol inhibits the synthesis of β-galactosidase, a critical enzyme used in the hydrolysis process.

Also, absorbance in the fourth treatment (containing E. coli, azide, and IPGT) as well as fifth treatment (containing glucose, IPGT, and E. coli) is significantly minimal. Sodium azide is known to block the last step of the electron transport chain hence stopping the cell from producing ATP. On the other hand, glucose is a major inhibitor of β-galactosidase. Thus, in the presence of glucose, E. coli will favorably metabolize glucose and not lactose. This is always the case until the glucose is completely depleted. Only then will the IPGT (β-galactoside) induce the production of β-galactosidase necessary for the hydrolysis of lactose into glucose and galactose (Görke & Stülke, 2008). Therefore, from the experiment, the only steady production of the ONP was observed in the second treatment (involving E. coli and IPGT). Primarily, the treatment has all the ideal conditions that necessitate the efficient production of the enzyme β-galactosidase. Principally, the procedures lack glucose and chloramphenicol (inhibitors) as well sodium azide (terminator).

The lack operon process can be enhanced or reduced by several mutations, primarily as it relates to the production of β-galactosidase. Majorly, mutations of the lac operon genes (lacA, lacZ, and lacY), can impact the production of the lactase enzymes (Mondin et al., 2002).

Conclusion

In summary, the lac operon is a useful operon necessary for the breakdown and transportation of lactose in several bacteria, including E.coli. It is an analytical technique used in the experiments involving gene control in bacteria. Even though most bacteria use glucose as the source of its carbon requirement, lac operon is an alternative in the absence of glucose as it enables the breakdown of lactose ("Khan Academy," 2016). The lac operon genes expressible are lacA, lacZ, and lacY. The process is possible when lactose is the important source of sugar in the bacterium. Mutations in these genes can either cause a reduced or increased production of the enzyme β-galactosidase.

• Measurement of β-galactosidase using ONPG
• Materials
  • • • Samples of E.coli on ice
      • O-nitrophenyl B-galactoside {ONPG} SOLUTION
      • 1M sodium carbonate
• Procedure

The tubes containing E.coli were placed in a water bath at 37c and left for 5 minutes to bring the contents to the working temperature of the assay. Afterward, 0.5ml of ONPG was added at 30 seconds interval to each tube and the contents mixed thoroughly. The tubes were then incubated for 15 minutes at room temperature. The reaction was stopped eventually, by the addition of 0.5ml of 1M sodium carbonate at 30 seconds intervals and the contents mixed ("Beta-Galactosidase Activity Assay," 2016). The spectrophotometer was set to read absorbance at 420nm. Time sample from each flask was used as the blank to zero the spectrophotometer. The absorbances of the incubated samples were read separately and recorded.

Results

The following results were obtained for β-galactosidase activity which was shown by its absorbance at 420nm after been incubated with ortho-nitrophenyl B-galactoside.

Time {minutes

Water+ E. coli

IPTG+E. coli

IPTG + E. coli + Cm at35 mins

E. coli + Azide + IPTG

glucose +E. coli + IPTG

0

0.000

0.00

0.00

0.00

0.00

15

0.000

0.072

0.116

0.005

0.005

30

0.000

0.020

0.217

0.00

0.009

45

0.000

0.285

0.257

0.018

0.009

60

0.000

0.385

0.274

0.015

0.013

Description

Treatment

Description

Treatment One (Water+ E. coli)

No activity at all.

Treatment Two (IPTG+E. coli)

A steady increase in the yellow colour with time demonstrated by the increasing absorbance.

Treatment Three (IPTG + E. coli + Cm at35 mins)

A steady increase in absorbance until the 35th minute.

Treatment Four (E. coli + Azide + IPTG)

Little activity.

Treatment Five (glucose +E. coli + IPTG)

Little activity.

Discussion

From the experiment, it is evident that the extent of the activity (measured by the amount of yellow color and absorbance) is directly proportional to the presence of the inducer (Isopropylthiogalactoside). This study is, therefore, a typical example of a lac operon. Thus, the amount of IPGT (β-galactoside) present reflects the production of the β-galactosidase enzyme necessary in the hydrolysis of lactose into galactose and glucose. Lack or inhibition of the inducer means the absence of the β-galactosidase enzyme hence a reduced production of the yellow product, o-nitrophenol (ONP).

For example, in the treatment involving water and E. coli only, there is significantly no activity observed because there is no inducer (IPGT) to trigger the production of β-galactosidase that is responsible for the hydrolysis process. Therefore, absorbance at all times remains at zero (see appendix one). Read More