Question

Detailed explanatio of Lac operon experiment, when the bacteria were placed in dropper bottles containing glycerol as a carbon source. 

Answer 1

• The ββ- galactosidase hydrolyses lactose i.e., ββ- galactosidase to galactose and glucose. 
• The permease is responsible for  the transport of lactose into the cell.
• The function of acetylase  is to catalyase the transfer of acetyl group from one molecule to another.
• The 3 structural genes Z, Y, and A transcribe into a single mRNA with independent translation units for the synthesis of distinct enzymes.
• An mRNA coding for more than one protein is known as Polycistronic mRNA.

Catabolite gene activator protein:

• The cells of E.coli utilize glucose in preference to lactose, when both of them are present in the medium.
• After the depletion of glucose in the medium, utilization of lactose starts.
• This indicates that glucose somehow interferes with the induction of lac operon, which is explained below:
• The attachment of RNA polymerase tom the promoter site requires the presence of a catabolite gene activator protein (CAP) bound to cyclic AMP.
• The presence of glucose lowers the intracellular concentration of cAMP by an unknown mechanism. 
• Due to diminished levels of cAMP, the formation of CAP-cAMP is low.
• Therefore, the binding of RNA polymerase to DNA and the transcription is almost negligible in the presence of glucose.
• Thus, glucose interferes with the expression of lac operon by depleting cAMP levels.
• Addition of exogenous cAMP is found to initiate the transcription of many inducible operons including lac operon.
• Therefore, it is very clear that the presence of CAP-cAMP is essential for the transcription of structural genes of lac operon.
• Therefore, CAP-cAMP acts as a positive regulator for the gene expression.
• As a result the lac operon is subjected to both positive and negative regulation.

                                                  

Incubation:

• The usual temperature in lab is 20 to 45⁰C.
• This controls the atmospheric gases such as oxygen and carbon dioxide.
• The temperature has to be adjusted according to the requirements (max, min, or intermediate).
• Oxygen and carbon dioxide requirements should be done.
• p^H should be maintained.
• Osmotic pressure has to be maintained.
• Hydrostatic pressure has to be maintained.

         As a final, the culture have to be incubated in the new media to reduce the risk of microorganisms that could be pathogenic to humans.

Lac operon

• In bacteria, the operon is the coordinated unit of genetic expression and is the functioning unit of DNA containing cluster of genes under the control of a single promoter.
• In 1961, Jacob and Monod introduced the concept of operon and based on their observations on the regulation of lactose metabolism in E.coli, it was popularly known as Lac operon.

Structure of Lac Operon:

• The lac operon consists of a regulatory gene (I ) for inhibition, operator gene (O), and 3 structural genes (Z,Y,A).
• Besides these genes there is a promoter site (P), next to the operator gene where the enzyme RNA polymerase binds.
• The structural genes Z, Y and A respectively code for the enzymes  ββ- galactosidase, galactoside permease and galactoside acetylase.