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Wize University Biochemistry Textbook > Genetic Engineering of Proteins

Basics of Genetic engineering

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Genetic Engineering

  • Allows us to identify the role of every amino acid in a protein
  • Used to study protein structure and function
  • Eg. Directed mutatgenesis
  • Common applications: over expressing proteins in genetically manipulated bacterial "factories" to produce high quantities of desired protein


https://commons.wikimedia.org/wiki/File:Central_dogma_of_molecular_biology.svg. Philippe Hupé. This file is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license.
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Genetic manipulation

Select genes can be input ("cloned") into plasmids either in their native form or manipulated/mutated form for genetic manipulation or protein analysis

Cloning

  • Genes can be cloned from any genomic DNA
  • These genes may be manipulated
  • There are 4 steps in protein cloning
  1. Clone the gene from cDNA or mRNA or in prokaryotes, genomic DNA
  2. Insert the cloned gene into an expression vector (plasmid)
  3. Insert this plasmid construct into an expression host (bacteria, typically E. coli)
  4. The bacteria will grow the protein for isolation

https://commons.wikimedia.org/wiki/File:Steps_of_Molecular_Cloning.png. Alexpicardal97. This file is licensed under the Creative Commons Attribution-Share Alike 4.0 International license.
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Plasmids (Expression Vectors)

  • Circular form of double stranded DNA
  • Often are or are derived from naturally occurring bacterial DNA
  • Insertion of a gene of interest into a plasmid results is recombinant DNA (DNA consisting of genetic material from several sources)
  • Contain key components:
  • Origin or replication
  • Selection marker
  • Multiple cloning site
  • Promoter
  • Circularized plasmids (with or without gene insert) can be replicated in bacteria in order to create more plasmid or protein (translated from gene insert)
https://commons.wikimedia.org/wiki/File:PBR322.svg. This file is ineligible for copyright and therefore in the public domain because it consists entirely of information that is common property and contains no original authorship.
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Restriction Enzymes

  • Restriction enzymes (restriction endonuclease) - RE - cut double stranded DNA at palindromic sites
  • REs originate form prokaryotic cells
  • Create "blunt" or "sticky" ends

5'.....C TCGAG.....3' 5'.....GCC GGC.....3'

3'.....GAGCT C.....5' 3'.....CGG CCG.....5'

XhoI NaeI

  • To insert a gene into a plasmid, you need to introduce RE sites to the ends of the gene (by PCR)
  • These sites must match RE sits in the plasmid of interest
  • Complementary sequences will be fused by DNA ligase

https://commons.wikimedia.org/wiki/File:Figure_17_01_06.png. This figure was adapted from CNX OpenStax's image licensed under the Creative Commons Attribution 4.0 International license.
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Polymerase Chain Reaction (PCR)

  • Used to amplify and alter genetic material using short complementary oligos called primers:
  • Genes for insert into plasmid

5'ATGCAGTCCGGGAATTG...................................................................................... 3'
3'.................................................................................TGCAACTTTGAACGTTTTAG 5'

  • Directed mutagenesis in a protein

5'ATGCAGTCC............................GGGAATTG.......................................................... 3'
3'...................................................CCCTTAAC..............................TGAACGTTTTAG 5'


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Steps in a PCR

  1. Denaturation: double-stranded DNA is separated at high temperatures
  2. Annealing: Primers bind to their complementary DNA at primer specific temperatures
  3. Elongation: nucleotide bases are added onto the ends of primers by thermostable DNA polymerase.
  4. Repeat for a designated number of cycles, each one doubling the number of genes present

https://commons.wikimedia.org/wiki/File:Polymerase_chain_reaction.svg. Enzoklop. This file is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license.


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Which of the following is an example of a palindromic sequence?

a) GGCTTA
CCGAAT
b) GCTTCG
CGAAGC
c) GATATC
CTATAG
d) CCCCAGGG
GGGGTCCC

c) To be palindromic a sequence has to have the same order of amino acids from 5' -> 3' in both strands
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Assuming you just want to amplify a WT (unaltered) gene, describe the gene cloning process from start to finish including the key components of plasmids, the role of restriction enzymes, and PCR in your answer.

A gene cloning workflow looks like:
  1. Identify the gene you wish to clone
  2. Chose the plasmid you wish to work with and select the restriction enzyme cut sites you will use when inserting the gene into the multiple cloning site
  3. Create primers for PCR amplification of the gene that are both complementary to the gene and contain the selected RE cut sites added on to the ends
  4. Perform PCR to amplify the gene from genomic DNA (prokaryotic) or cDNA (eukaryotic)
  5. Digest both the plasmid and the PCR product to create complementary "sticky" or "blunt" ends
  6. Ligate the gene into the plasmid using DNA ligase
  7. The plasmid should contain an origin of replication (where DNA replication is initiated leading to the creation of more copies of the plasmid), a promoter (where RNA polymerase binds allowing for the transcription and translation of the protein of interest), and a selection marker (allows for the bacterial expression host to be selected for via antibiotics)
  8. The complete plasmid is then transformed into a bacterial expression host where the protein will be amplified and await extraction and purification
What is NOT a common feature found in in every plasmid?