Iptg induction

Editor's Notes

• #3 (an enzyme which hydrolyses lactose into glucose and galactose), (an enzyme which promotes lactose transport across the cell membrane of bacterium) (an enzyme which transfers acetyl groups from acetyl-coenzyme A to B--galactoside),
• #4 This reaction can be conveniently followed in the laboratory through the use of alternative galactoside substrates that form colored products such as X-Gal The genes encoding the LacI repressor are actually located upstream of the Lac Operon. The LacI gene is not regulated; therefore, it is produced continuously. It binds to the Lac Operon in the promoter region; however, it does not bind if there is lactose in the cell. Well, the cell produces very low levels of β-galactosidase even when not in the presence of lactose. In these very low lactose conditions, β-galactosidase has a different function: it cleaves lactose and recombines it to form allolactose, which acts as an inducer for LacI. It binds to LacI and causes a conformational change, which in turn makes LacI unable to bind to the promoter region of the Lac Operon.
• #5 A pair of the amino-terminal domains come together to form the functional DNA-binding unit. The lac repressor binds DNA by inserting an α helix into the major groove of DNA and making a series of contacts with the edges of the base pairs as well as with the phosphodiester backbone The DNA-binding site of the Lac repressor is able to bind with high affinity to only one DNA sequence in the entire E. coli genome—the lac operator. The specificity of high-affinity DNA binding ensures that the repressor will bind only to the site on the DNA near the genes that it is controlling and not to random sites distributed throughout the chromosome. By binding to the operator, the repressor prevents transcription by RNA polymerase that has bound to its lac promoter site. The lac repressor binds 4 × 106 times as strongly to operator DNA as it does to random sites in the genome. This high degree of selectivity allows the repressor to find the operator efficiently even with a large excess (4.6 ×106) of other sites within the E. coli genome
• #8 Molecules that induce enzyme synthesis but are not metabolised themselves are called gratuitous inducers. IPTG is a gratuitous inducer of lac opero.
• #10 such as alpha-select, CH3-Blue and BIOBlue cells Storage conditions (working solution): Stock solutions of the product in water (e.g., 1 M IPTG) stored at -15 to -25 °C, are stable up to six months. Recommendations for use  Dissolve to desired concentration in Water, 18.2MΩ (DNase/RNase-free).  Filter sterilize through a 0.22 micron disposable filter.  Store solution at -20 °C.  For blue/white screening, 0.1mM final IPTG concentration in LB (Luria Broth) media is recommended
• #11 POSITIVE REGULATION: CAP-cAMP ComplexRemember from before, the absence of the LacI repressor is not the only factor that allows transcription to occur. There is also a form of positive regulation that occurs via the CAP-cAMP Complex, the formation of which is controlled by the levels of glucose within the cell. As glucose levels in the cell begin to decline, E. Coli responds by beginning to synthesize cyclic adenosine monophosphate, or cAMP. As cAMP concentration increases, it binds to a catabolite activator protein, or CAP. cAMP acts as an inducer for CAP, causing a conformational change that allows CAP to bind to the promoter region of the Lac Operon. This cAMP-CAP complex interacts with the RNA polymerase, increasing its affinity for the Lac promoter. Without attachment of the cAMP-CAP complex, or in high levels of glucose, affinity wouldn't be high enough to cause significant transcription. CAP protein binds as a dimer to an inverted repeat that is at the position -61 relative to the start site of transcription. The CAP binding site on DNA is adjacent to the position at which RNA polymerase binds.
• #12 Over the last few decades, a major effort has been made to engineer the lac promoter, lac proteins, and several accessory proteins, in order to optimize lac-dependent expression and increase the flexibility and functionality of the system. One disadvantage of the lac promoter for mediating protein expression is that its promoter sequence TTTACA (–35) and TATGTT (–10) differs in two bases from the canonical E. coli promoter sequence TTGACA (–35) and TATATT (–10). In particular, the G to T change in the –35 region is deleterious to the activity of the promoter (2). To alleviate this problem, a promoter has been engineered that bears the –35 region from the trp (tryptophan) promoter and the –10 region and the operator from lacUV5. There are two forms of the trp/lac promoter, trc and tac, which give rise to similar levels of protein expression (12,13). The increased activity of trc and tac requires that LacIq or overexpressed LacI be present to suppress transcription in the absence of induction. LacIq is usually expressed from the F' element or, alternatively, from a derivative of the pLysS/E or Rosetta™ helper plasmids
• #17 1The galactoside substrate X-Gal produces a colored product on cleavage by β-galactosidase. The appearance of this colored product provides a convenient means for monitoring the amount of the enzyme both in vitro and in vivo. X gal to 5,5 dibromo 4,4 dichloro indigo