Investigation of Induction Time of lac operon in E. Coli with IPTG, and Comparision of the Amount Beta-galactosidase produced with Lactose, IPTG and Antibiotic.
An operon is a group of genes that are arranged side by side with a regulatory gene. It also includes a promoter gene, operator gene, structural genes. Regulatory genes control transcription with positive or negative signal. (Jacob, and Monod, 1961) A positive signal, inducer, would stimulate binding of RNA polymerase by binding to the operator gene and transcription would occur. A negative signal, repressor, would not have any transcription occurring as it would not allow RNA polymerase to bind to promoter. This control of gene expression allows organism to not waste energy producing enzymes not needed. (Murray, 2012)
The lac operon has a promoter, operator, regulatory and structural genes. The structural genes consists of the lacZ, lacY and lacA gene which codes for enzymes beta-galactosidase, permease and thiogalactoside transacetylase respectively. (Campbell and Farrell, 2009) Lactose is dissacharide with galactose and glucose binded together by a glycosidic linkage. Beta-galactosidase hydrolyses that linkage, hence breaks down lactose to be used as a carbon source. (Campbell et al, 2009) Permease is a membrane-transport protein, it goes against the membrane potential concentration gradient to drive lactose into cell. (Kaback, Sahin-Toth and Weinglass, 2001) The function of thiogalactoside transacetylase is still being studied but there seems to be a connection defending the cell against antibiotics. (Andrews and Lin, 1976) The regulatory gene is called the lacI gene, it produces the repressor which causes the negative control for gene expression. (Murray, 2012)
The lac operon transcription occurs only when there is no repressor bound to the operator and when the cAMP.CAP complex is bound to the promoter. In levels of high glucose, cyclic AMP (cAMP) levels are low hence no cAMP.CAP complex produce to continue transcription. In absence of glucose, cAMP levels are high, this results in formation of cAMP.CAP complexes that binds to promoter region and attracts RNA polymerase to bind to promoter for transcription. (Campbell et al, 2009) This control mechanism is called catabolite repression and is a positive control. (Murray, 2012)
In presence of an inducer such as allolactose or isopropyl beta-D-1-thiogalactopyranoside(IPTG), the repressor and inducer binds together and becomes inactive and cannot be bound to operator anymore. (Campbell et al, 2009) Without any repressor bound RNA polymerase can continue with transcription of genes. The allolactose is a derivative of lactose and IPTG is a synthetic analogue. (Galea and Murray, 2012) Without an inducer, repressor is bound to operator and blocks transcription from occurring. This control is a negative one by the repressor. (Murray, 2012)
The binding RNA polymerase to promoter is weak so not only must there be no repressor blocking the promoter region, the cAMP.CAP complex must be bound to promoter for transcription to occur. (Campbell et al, 2009) In presence of glucose and in absence of inducer, no transcription would occur. (Murray, 2012)
Besides controlling of gene expression, antibiotics can be used to inhibit either transcription or translation. In this experiment, chloramphenicol, rifampicin and streptomycin are used. Chloramphenicol and streptomycin inhibits ribosome function, no protein synthesis can occur as it is inhibiting at the translation phase. (Neu and Gootz, 1996) Rifamicin inhibits DNA directed DNA polymerase, no transcription can occur. (Neu et al, 1996) The effects of inhibition at translation or transcription are observed.
In this experiment, induction time of lac operon will be determined by incubating Escherichia coli(E.coli) and IPTG for different time points and assaying its beta-galactosidase activity. IPTG is...