Transcription is a process by which a segment of DNA is copied into a complementary sequence of RNA that is used to be translated into proteins. Transcription involves promoters to which RNA polymerase binds, isomerization, elongation, and termination. These processes are regulated by binding proteins. Many factors influence transcription productivity including DNA supercoiling. There are two types of supercoiling, positive and negative. Positive supercoiling occurs when the right-handed double helix of DNA becomes twisted more tightly and begins to knot or deform. The negative supercoil is twisted into a left-handed conformation and is usually the supercoil present in the DNA of most organisms. In the literature, supercoiling has been studied at different promoters on a plasmid using a series of topoisomers in order to discover how much supercoiling affects transcription and how it is affected. In the experiment, a plasmid called pSA850 was used to study how it affects transcription. This plasmid is composed of multiple promoters followed by a transcription terminator that produces sequences of different lengths. Some promoters present on the plasmid included lacP, galP1 and galP2, pP, bP, and rP. To prepare topoisomers of plasmid pSA850, it was incubated in a mixture of calf thymus topoisomerase 1 and ethidium bromide. The reactions ended and the proteins as well as ethidium bromide were removed by a phenol-chloroform extraction and subsequently isoamyl alcohol was also used twice to extract. TAE-buffered agarose gel containing chloroquine was used to measure the average linkage number difference of produced topoisomers. The link number was determined to be 332 using 10.5 base pairs per turn, and the superhelical densities were also concluded. In vitro transcription was initiated in a mixture including DNA template, RNAP, Tris-acetate, pH 7.5, magnesium acetate, potassium glutamate, and rRNasin. The initial mixture was incubated for approximately five minutes and then added to a mixture of NTPs containing ATP, GTP, CTP, UTP, and [α-32P]UTP for the reactions to begin and then incubated. The reactions were then terminated using BRL which is a STOP solution. The mixtures were heated for two minutes and to observe the resulting transcripts and abandoned molecules prepared from the promoters, 3 microliters of each sample was placed on polyacrylamide-urea sequencing gels. The gene regulatory proteins were present in varying degrees and therefore could determine whether different levels of topoisomers showed different effects.