Bacterial Cloning Process Bacterium Bacterial chromosome Plasmid Gene inserted into plasmid Cell containing gene of interest Gene of interest DNA of chromosome Recombinant DNA (plasmid) Plasmid put into bacterial cell Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Protein expressed by gene of interest Protein harvested Gene of interest Copies of gene Basic research on gene Basic research on protein Basic research and various applications Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth
Restriction Enzymes & Sticky ends Restriction enzymes are special enzymes, found in bacteria, that cut DNA at special places. The specific place that the DNA is cut is called a restriction site. When the restriction enzyme cuts the DNA at the restriction site, it creates fragments of DNA called restriction fragments. Restriction fragments have “sticky ends” that can match up with the ends of other fragments.
Cut the DNA and Bacterial Plasmid The first step in bacterial cloning is to use specific restriction enzymes to cut the DNA and bacterial plasmid. This will cut both at the same place! Once the DNA has been cut, you can mix the fragments together and allow them to be joined using ligase.
Reintroduce the Plasmid The next step is to reintroduce the recombined plasmid back into the bacteria. This is done through transformation bacteria picking up foreign DNA. The new bacteria are called a cloning vector. A vector is an organism that carries a recombined plasmid.
Let the Bacteria grow and reproduce You will know let the bacteria grow and reproduce. The recombined bacteria will grow through binary fission and create new bacteria that should have the plasmid.
Identify the transformed bacteria and culture the experimental bacteria Once you have grown the recombined bacteria, you will now need to isolate the experimental bacteria. 1. You will create a radioactive nucleic acid probe using phosphorous. 2. Then you will denature the DNA using heat to expose the bases. 3. The radioactive probe will join with the complimentary bases on the gene of interest. 4. Use a special film that will show the radioactive colonies and separate these from the others.
Source of the DNA for Transformation? Scientists must go from mRNA back to DNA to make the process easier. This is a tough thing to do because: -Prokaryotic DNA does not have introns. -Modified mRNA must be collected after it leaves the nucleus and turned back into DNA. -Use reverse transcriptase to turn single stranded RNA into double stranded DNA. You will need to first add a promotor sequence. -New cDNA (complimentary DNA) can be stored for use.