Question

3. I use the lac-T7expression system (what we're using in this lab) Normalized OD to express Cyt P4so in bacterial cells. I have two isolates, A and B Stran Glucose Lactose PT that I assume contain the Cyt Paso expressing plasmid. I makeA08318912 0825 total protein extracts and measure the absorbance at 418nm of B0.8760.882 0,799 the two isolates, grown in either Glucose, Lactose or IPTG. My results are shown in the table. 4. lusethe lac-T7expression system (what we're using in this lab) Carbon source Normaized oo.. 418 to express Cyt Paso in bacterial cells. I grow cells in either Glucose, Lactose or IPTG, and then purify Cyt P45o from these cells. The absorbance readings at 418nm for cells grown on different Carbon sources is shown. Glu Lac PTG 1398 9.447 16.214

Why is IPTG showing a higher OD418 reading when a carbon source version is present versus when IPTG is made as a protein extract?

Answer 1

1. IPTG is a molecular mimic of all-lactose, which is a metabolite responsible for starting transcription of the lac operon. So it is used as a inducer. IPTG binds to the lac repressor and releases the tetrameric repressor from the lac operator, thus allowing the transcription of the genes in the lac operon. But it cannot be metabolised. The sulphur atom create a chemical bond, which the cell can't hydrolyse. Thus the cell can not metabolise or degrade it and its concentration remains constant and the expression of the lac processor controlled genes won't be inhibited during the experiment. This is why IPTG shows a higher OD ant 418 nm than the carbon sources like lactose or glucose.

2. The concentration of any particular protein would be higher in purified enzymes than total protein in the same volume. Thats why the absorbance, which is a measure of concentration, is showing higher values in purified products when compared with total protein concentration at OD418