Fermentation

Plasmid is a double stranded, circular, extra chromosomal DNA of bacterium. Size of plasmids range from 1-1000 k base pairs. They are very small as compared to chromosomal DNA which consists of millions of base pairs. It is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA. Plasmid can be transferred between same species or between different species. Many naturally occurring plasmids contain genes that provide some benefit to the host cells. In molecular cloning, plasmids are used as very important tool because they are used as vectors, which means that they’re useful in propagating foreign genes. In DNA cloning, recombinant DNA molecules are formed by inserting DNA fragments of interest into vector DNA molecules. The recombinant DNA molecules are then introduced into host cells, where they replicate, producing large numbers of recombinant DNA molecules that include the fragment of DNA originally linked to the vector.

Plasmid DNA which exists naturally in supercoiled form has more condensed structure than the DNA which is not coiled and is in unwounded or relaxed form. DNA gyrase is responsible for the supercoiling in the cell. Supercoiling is important biologically as very large DNA molecules can fit in the cell only if they are supercoiled. Supercoiling also influence the gene expression. There are other forms of plasmid DNA molecules which are linear, dimer, trimer, Nicked circles but the supercoiled form has the fastest migration rate out of all forms. In this experiment, first suspended bacterial cells are mixed with RNase and cell lysis solution. Cell membrane is dissolved and protein is denatured because of the presence of sodium dodecylsulfate detergent. The pH is very high because of sodium hydroxide. Potassium acetate neutralization buffer is added to neutralize the alkaline condition created by sodium hydroxide. Potassium acetate precipitates the SDS with its membrane fragments and proteins. Centrifugation of these complexes removes most of the chromosomal DNA which is trapped inside. Isopropanol is added to precipitate the plasmid and rest of the RNA. To resuspend the DNA precipitate, Tris buffer is used, this contains RNASe, which degraded RNA. Gel loading solution in the concentrated form prepares the sample for electrophoresis by making it denser than the buffer is used for electrophoresis. In this experiment, extraction of 3000 base pair plasmid is done from E.coli cells. (Kuhn,2014).

Fermentation in yeast

Table below shows the measurements taken: