Gel Electrophoresis (a lab technique used by scientists to measure the size of DNA fragments) and recombinant plasmids

Process of Gel Electrophoresis

1. DNA samples are placed in the wells at one end of the gel

a standard ladder of DNA fragments with known sizes are typically loaded into one well, which is required for estimating the size of any unknown DNA fragments by comparing them to the known fragments in the standard ladder

the agarose is immersed in a buffer solution that helps carry the electric current

2. an electric current is passed through the gel using two electrodes (one pos, one neg)

since DNA is negatively charged (due to the phosphate backbone), it is attracted to the positive electrode

when the electrical current is applied, DNA fragments will move from the wells through the pores in the gel and towards the positive electrode

3. smaller DNA fragments move faster through the gel and therefore travel further than larger fragments which don't move as easily through the gel

4. DNA is difficult to see with the naked eye so the gel is stained with a fluorescent dye, allowing the bands of DNA to be visualised under a UV lamp

Materials for making a recombinant plasmid

• gene of interest

• plasmid vector

• restriction endonuclease

• DNA ligase
  
  

Gene of Interest

a sequence of DNA of a particular protein is isolated using restriction endonucleases and amplified using the polymerase chain reaction

• gene of interest must not have introns when it is inserted into the plasmid as prokaryotes don't do RNA processing (so they wouldn't know what to do with them)

Plasmid vector

a plasmid vector is selected in which the gene of interest will be inserted in

most plasmid vectors contain

• restriction endonuclease recognition sites, so the plasmid can be recognised and cut by a restriction endonuclease which allows the gene of interest to be inserted

• antibiotic resistance genes, to confer resistance to antibiotics

• ‘origin of replication’ a sequence that signals the start site for DNA replication in bacteria

• ‘reporter gene’ a gene with an easily identifiable phenotype that is used to identify whether the plasmid has taken up the gene of interest

Restriction endonuclease

the gene of interest and the plasmid are both cut with the same restriction endonuclease to generate identical sticky ends

• the sticky ends of the gene of interest will be complementary to the sticky ends on the plasmid vector, which allows them to easily form hydrogen bonds

• (blunt end restriction enzymes may also be used but are less targeted as they can bond with any other blunt end)

DNA ligase

DNA ligase is added to join the gene of interest to the plasmid vector by forming phosphodiester bonds, creating a circular piece of DNA, called a ‘recombinant plasmid’

Bacterial transformation

the process of inserting a recombinant plasmid into the cytoplasm in bacteria

• two primary methods; heatshock, electroporation

Insulin

an important hormone responsible for regulating blood glucose levels

• the insulin protein has a quaternary structure consisting of two polypeptide chains, known as alpha and beta subunits

• this means to produce insulin, two different recombinant plasmids and therefore two different transformed bacteria samples (one producing each subunit)

these two polypeptide chains must fold individually and then be joined together by a disulphide bridge

Process of making recombinant plasmids

1. the gene of interest and the plasmid are both cut with the same restriction endonuclease to generate identical sticky ends

2. DNA ligase is added to join the gene of interest to the plasmid vector by forming phosphodiester bonds, creating a circular piece of DNA, called a ‘recombinant plasmid’

3. inserting a recombinant plasmid into the cytoplasm in bacteria

   • two primary methods; heatshock, electroporation