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Techniques For The Investigation Of Lac Operons

624 words - 3 pages

The micro-organism used in this lab was E.coli, which is a prokaryote. In general, prokaryotes are small, compact genomes that have a single, round DNA molecule. The genes are tightly packed, with almost no space between them. In the case of E.coli, it has thrA, 1 nucleotide, thrB, thr C.

Enzymes of a single biochemical pathway are coded by the operon: introns are absent, so all genes are not interrupted and the sequence is non-repetitive. An operon is a group of adjacent genes which are transcribed as a unit into a single polycistronic mRNA molecule. The lac operon is necessary for the transportation and metabolism of lactose.

An operon is made up of three features: promoter, operator and structural genes. The promoter site is where the RNA polymerase (Plac) is attached. The operator is the site where repressor protein binds, blocking RNA polymerase from binding, which in turn stops the transcription of the operon. Lastly, there are the structural genes, which are numerous genes, which code for the enzymes of a metabolic pathway, transcribing as a unit.

There are two techniques which may be used to investigate the induction of the lac operon. The first is using IPTG to induce the lac operon and then ONPG and B-galactosidase, which will hydrolyse the ONPG to produce galactose and nitrophenol (can be measured with the use of a spectrophotometer). The second technique is to hydrolyse x-gal using the ability of B-galactosidase. Induced and uninduced E.coli cultures are plated and the colours of the colonies are examined.

Protocol
Two bottles containing 10ml broth cultures of E.coli, labelled “induced” and “uninduced” were used. Then, after adding 100ml of IPTG to the induced culture, it was quickly mixed and 1 ml was transferred into a microfuge tube (labelled as 0 mins induced). Next, 1ml...

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