20 The first recombinant DNA plasmid that combined two dif f

20. The first recombinant DNA plasmid that combined two dif ferent biological activities from different sources and was readily transferred into bacteria was reported in 1973 b the research groups of Stanley Cohen and Herbert Boyer. In this work, the investigators began with the large, natu- rally occurring circular plasmid R6-5 (~98,500 bp), which included genes conferring resistance to multiple antibiot- ics, including tetracycline and kanamycin. Prior to their study, R6-5 had been sheared into fragments, the fragments used to transform E. coli, and tetracycline-resistant colonies selected. A smaller circular plasmid had arisen from that work, pSC101 9000 bp), which replicated normally and conferred only the tetracycline resistance. The researchers assumed that the smaller plasmid represented a piece of the larger one that had ligated into a smaller circle, which included a replication origin and the tetracycline-resistance gene. A third plasmid had also been generated when plas- mid R6-5 was cleaved with EcoRI, the fragments used to transform E. coli, and cells selected for growth on kanamy- cin. This third plasmid was called pSC102 27000 bp), and it did not confer resistance to tetracycline. The ultimate objective was to combine pSC101 with fragments from R6-5 or pSC102 to generate a new plasmid that conferred a demonstrable new biological activity. The investigators first cleaved all three plasmids with EcoRI. The cleavage products were subjected to electrophoresis on an agarose gel, and the DNA fragments were visualized by staining with ethidium bromide (a fluorescent mole- cule that binds to DNA by intercalating between adjacent base pairs). This generated the pattern in Figure 1. Elec- trophoresis proceeded left to right, as shown in the figure, so the bands decrease in size from left to right (ane a is psc102, lane b, R6-5; lane c, pSC101)

Solution

Given in the problem:

large naturally occurring circular plasmid

R6-5 had been sheared into fragments, the fragments used to transform E.coli, and tetracycline resistant colonies selected. small circular plasmid

When R 6-5 was cleaved with ECoRI, fragments transformed in to E.coli

Name of the plasmid

R6-5      

pSC101

pSC102

Base pairs

98,500

9000

27000

Antibiotic resistance

Multiple:

Tetracycline and Kanamycin

The smaller plasmid represents a piece of R 6-5 (larger part) and that was ligated into a circle with replication origin and tetracycline resistance gene.

have only kanamycin resistance.

Objective:

To combine pSC101 with R6-5 fragments or pSC 102 to generate new plasmid.

The investigators cleaved all 3 plasmids with EcoRI., then cleaved products were electrophoresed on agarose gel, DNA fragments were visualized by EtBr stain.

Explaining the diagram:

Lane a

pSC 102

27000bp

3 different bands appeared

Lane b

R6-5

98500bp

Many bands appeared because it will have origin of replication, Multiple Antibiotic resistance sites like Tetracycline and Kanamycin

Lane c

pSC 101

9000bp

1 single band

Answer : There were two similar sized fragments generated by digestion with the enzyme. So, that the amount of DNA of that size will be double the amount (number) of other sized fragments. Hence the band in question seem to appear with double the intensity.

Given in the problem:	large naturally occurring circular plasmid	R6-5 had been sheared into fragments, the fragments used to transform E.coli, and tetracycline resistant colonies selected. small circular plasmid	When R 6-5 was cleaved with ECoRI, fragments transformed in to E.coli
Name of the plasmid	R6-5	pSC101	pSC102
Base pairs	98,500	9000	27000
Antibiotic resistance	Multiple: Tetracycline and Kanamycin	The smaller plasmid represents a piece of R 6-5 (larger part) and that was ligated into a circle with replication origin and tetracycline resistance gene.	have only kanamycin resistance.