P-Glo You should have downloaded the PGlo handout. Work in groups of four.
The pGLO plasmid is an engineered plasmid used in biotechnology as a vector for creating genetically modified organisms. The plasmid contains several reporter genes, most notably for the green fluorescent protein (GFP) and the ampicillin resistance gene. GFP was isolated from the jelly fish Aequorea victoria. Because it shares a bidirectional promoter with a gene for metabolizing arabinose, the GFP gene is only expressed in the presence of arabinose, which makes the transgenic organism fluoresce under UV light. GFP can be induced in bacteria containing the pGLO plasmid by growing them on +arabinose plates.
pGLO is made up of three genes that are joined together using recombinant DNA technology. They are as follows: -Bla, which codes for the enzyme beta-lactamase giving the transformed bacteria resistance to the beta-lactam family of antibiotics (such as of the penicillin family) -araC, a promoter region that regulates the expression of GFP (specifically, the GFP gene will be expressed only in the presence of arabinose) -GFP, the green fluorescent protein
P-Glo is a transformation exercise. We briefly talked about plasmids and transformation in lecture. Some organisms have the ability to take up DNA from their environment. We will use E. coli and force it to take up DNA in the form of a plasmid. In E. coli, there is a promoter region for ampicillin resistance. Another promoter region contains the gene for the sugar, arabinose (inducible operon). E. coli requires arabinose to turn the gene off.
We have a plasmid which contains the gene from a jellyfish that allows the organism to glow under florescent light. We are going to introduce the gene into the E. coli, and grow them in plates (LB instead of TSA). Two plates will have E. coli which are ampicillin resistant, two plates will have E. coli which are not ampicillin resistant. One of each of those plates will have E. coli which has had the plasmid inserted, the other will not. One of the other two plates (positive; contains the plasmid) will contain arabinose, the other will not. We will be incubating in the presence of ampicillin, and that will select for those organisms which can take up the plasmid. We will also be incubating for the presence of the sugar, arabinose, because it is required to turn the gene on. If there is no gene for ampicillin resistance or arabinose, it should not be able to glow.
Take one colony and place it in one of each of the pink and blue tubes. The technician will put plasmid in the pink tube. Put both of the tubes into ice for 10 minutes to force the transformation. Each tube also contains calcium chloride, which pokes holes in the outer membrane, allowing the DNA to gain access to the inside of the cell. Now we have to shock the cell to cause the DNA to be taken up. To do this, put them into the water bath at 57 degrees for 50 seconds, then back on ice for 2 minutes. Then collect your tubes and add 250 µl of LB broth to both tubes and incubate at room temperature for 10 mins. This allows for replication of the cell and incorporation of the plasmid. Then pipette 100 µl from the blue tube to the two negative plates (contain no plasmid; one has arabinose and one does not), and pipette 100 µl from the pink tube to the two positive plates (contains plasmid; one has arabinose and one does not). Next lab, we will expose them to UV light to excite the proteins and see which ones show green fluorescence. The only plate that should glow is the one with the plasmid plus arabinose.
Plates LB/amp positive (has plasmid) LB/amp/arabinose; positive LB/amp negative (before transformation, they have no amp resistance. Should not glow) LB negative (just a media like TSA; will grow, but no glow)
The promoter region (can turn off and on; is an operon. It is turned off and on by arabinose). The non-transformed cells do not have the ampicillin resistance gene.