Instructors: M/WGerry ProdyCB 444 Scott Delbec T/RClint SpiegelCB 443 Peter Littlefield

Course Overview Lab Notebooks Prelab/Research Project/Paper Lab Etiquette Background: Everything in 471 (and 2) is fair game.

Figure 5-43The pUC18 cloning vector. Page 106

Figure 5-46Construction of a recombinant DNA molecule. Page 108

N-terminal 6xHis tag; TEV protease cleavable pET27 derivative T7lac promoter 5’ cloning site: BamHI low copy plasmid Kan-resistant MGSS(H6)ENLYFQ GS-protein ORF TEVprotease cleavage site BamHI cloning site

Figure 31-1The induction kinetics of  -galactosidase in E. coli. Page 1217

The lac operon E-coli uses three enzymes to take up and metabolize lactose. The genes that code for these three enzymes are clustered on a single operon – the lac Operon. What’s lactose??

Figure 31-2Genetic map of the E. coli lac operon. Page 1218

The lac repressor gene Prior to these three genes is an operator region that is responsible for turning these genes on and off. When there is not lactose, the gene for the lac repressor switches off the operon by binding to the operator region. A bacterium’s prime source of food is glucose. So if glucose and lactose are around, the bacterium wants to turn off lactose metabolism in favor of glucose metabolism.

Isopropyl thio -  -D- galactoside

Induction. Allolactose is an isomer formed from lactose that derepresses the operon by inactivating the repressor, Thus turning on the enzymes for lactose metabolism.

The lac operon in action. When lactose is present, it acts as an inducer of the operon (turns it on). It enters the cell and binds to the Lac repressor, causing a shape change that so the repressor falls off. Now the RNA polymerase is free to move along the DNA and RNA can be made from the three genes. Lactose can now be metabolized (broken down).

When the inducer (lactose) is removed The repressor returns to its original shape and binds to the DNA, so that RNA polymerase can no longer get past the promoter. No RNA and no protein is made. Note that RNA polymerase can still bind to the promoter though it is unable to move past it. That means that when the cell is ready to use the operon, RNA polymerase is already there and waiting to begin transcription.

Lac movie Lac and trp

Expression tutorial

Figure 31-25The base sequence of the lac operator. Page 1239

Figure 31-37aX-ray structure of the lac repressor-DNA complex. Page 1249

Lac repressor binding to DNA animation ex.htm