The document discusses two important plasmids in recombinant genetics: pBR322 and pUC8. pBR322, about 4300 bp long, contains antibiotic resistance genes, while pUC8 is derived from pBR322 and allows bacteria to utilize lactose. A method using x-gal and IPTG allows differentiation of bacteria with recombinant plasmids from those with non-recombinant plasmids based on color changes, enhancing the effectiveness of genetic engineering.

1. Plasmids - pBR322 and pUC8
pBR322 Plasmid
One of the first plasmids to be used in recombinant genetics was called pBR322. It is
approximately 4300 bp in length and has two antibiotic resistance genes: Ap (Ampicillin)
and Tc (Tetracycline). Bacteria cells that are successfully transformed with this plasmid
are able to grow in the presence of both ampicillin and tetracycline antibiotics.

2. pUC8 Plasmid
The pUC8 plasmid was designed by scientists and contains the lac z gene. To produce
the plasmid, the pBR322 plasmid was cut in half with EcoR I and the section containing
the ampicillin resistance gene was combined with a DNA fragment containing the lac z
gene. As a result, the plasmid provides a transformed cell with both ampicillin resistance
and the ability to utilize lactose as a food source, since the lac z gene produces B-
galactosidase (degrades lactose).
Since the pUC8 plasmid was produced using EcoR I, this enzyme cannot be used to
insert desired genes when producing recombinant plasmids for use in genetic
engineering.
This plasmid was originally used in conjunction with a special mutant strain of E. coli
called JM101, which has a mutation in its chromosomal lac z gene and cannot
survive on media containing only lactose as a food source. If JM1010 bacteria
were successfully transformed with the pUC8 plasmid, they gained the ability to
use lactose as a food source. Therefore, only successfully transformed bacteria
would survive when grown on media containing only lactose and ampicillin.

3. Improving the use of pUC8
While it was possible to isolate the bacteria transformed with pUC8 on lactose and
ampicillin containing media, a problem existed. During the process of producing
recombinant pUC8 plasmids with additional genes of interest inserted in them, some
plasmids did not successfully incorporate the desired gene and closed back up into non-
recombinant plasmids. When bacteria cells were exposed to the mixture of plasmids, the
non-recombinant plasmids were taken into cells along with the recombinant
plasmids. Unfortunately, the non-recombinant plasmids also gave cells the ability to
metabolize lactose and survive in the presence of ampicillin.
Non-Transformed
No Plasmid
Dies from Ampicillin
and lack of Food
The solution to the problem of separating the JM101 cells transformed with recombinant
plasmids from the JM101 cells transformed with non-recombinant plasmids was solved
using the chemicals called X-Gal and IPTG. IPTG acts as an inducer that causes B-
galactosidase to bind to X-Gal. When X-Gal is broken down, it turns blue in color. Thus,
all the JM101 bacteria that had the plasmid with an active lac z gene (producing B-
Galactosidase) would appear blue. The key to using this was to insert the additional
desired gene in a location that would disrupt the lac z gene in the plasmid and use media
containing lactose, ampicillin, and other nutrients. By doing so, all the bacteria with the
recombinant plasmid would appear white and all the bacteria with the non-recombinant
plasmid would appear blue. The non-transformed bacteria would be killed by the
antibiotic and fail to grow. An additional benefit was that scientists were not restricted to
using E. coli from strain JM101. The technique could be successfully employed with any
type of bacteria cells that was used.
Non-Transformed
Transformed
Non-Recombinant Plasmids
Survives –does not produce
desired protein
Transformed
Recombinant Plasmids
Survives - produces desired
protein
Transformed
Non-Recombinant Plasmids
Blue in Color
(X-Gal is Degraded)
Transformed
Recombinant Plasmids
White in Color
(X-Gal is not Degraded)
Die