BIOL1007 Lecture Notes - Lecture 18: Molecular Cloning, Saccharomyces Cerevisiae, Puc19

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19 May 2018
Department
Course
Professor
Traditional biotechnology
Used to preserve foods, or make alcohol
-
Early fermentations used mixed cultures of naturally occurring bacteria and fungi
-
e.g. Scandinavian fermented fish storage unit c.7000 BC
-
Earliest firm evidence for alcoholic fermentation around 7000 BC in China, analysis of jars
revealed traces of alcohol and plants used for fermentation
-
Relationship with fermenting yeasts is ancient, and remains very strong
-
e.g. fermentation
Cellular
Molecular
e.g. fermentation
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Need some biology skills (esp. microbiology)
-
Don't need understanding of DNA, RNA,
proteins
-
Modern methods
-
Need high-level biology skills (esp.
microbiology)
-
Need understanding of DNA, RNA, proteins
-
Explain why microbes are useful for biotechnology, using examples
of specific fungi (S.cerevisiae) and bacteria (E.coli)
Microbes useful for biotech
Archaea
Source of thermostable polymerase enzymes for copying DNA sequences
Bacteria
Excellent hosts for cloning DNA and expressing proteins
Algae
Conversion of CO2+ light into biofuels (ethanol, H2)
Fungi
Excellent cloning and overexpression hosts
-
Yeasts
Antibiotic synthesis
-
Moulds
Contains machinery for biosynthesis of high-value products form simple raw materials
-
Screen clipping taken: 12/10/2017 3:22 PM
Hosts cells for biotech
Bacteria
E.coli
Fastest growth
-
Very easy to extract or add plasmid DNA
-
L18 - cell factories and biotechnology
Thursday, 12 October 2017
8:26 AM
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Very easy to extract or add plasmid DNA
-
Yeast
Saccharomyces
Better for cloning and expressing eukaryote genes
-
Generally recognised as safe (GRAS), can't cause infection
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Host cell factory needs instructions (ie. DNA) or blueprint to tell it which products to make
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Circular DNA elements found in microbes
Replicate independently of chromosomes
Plasmids most commonly used vector for delivery of foreign DNA into target host cell
Viruses can be used as vectors
e.g. pUC19 is a tame lab plasmid (has simpler structure compared to wild plasmid)
"wild" plasmids found in nature to allow microbes to swap useful genes
Plasmid-borne genes not essential, but useful under some specific conditions
E.g. antibiotic resistance genes
Where from?
Selectable marker - enables us to force cells to take up plasmid
Cloning site - add foreign genes here
Replication functions - ensures persistence in host
Key features making plasmids useful for biotech
Instructions delivered via PLASMIDS
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Explain what a plasmid is, and define the roles of different kinds of
plasmids in nature and in biotechnology
Create identical organisms OR make copies of a bit of DNA by adding it into a plasmid,
then replicating plasmid
Relies on enzymes for DNA cloning
Cloning - to make many copies of a biological entity
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Define the terms “DNA cloning”, “recombinant DNA”, “GMO”
Copying DNA needs thermostable polymerase
Cutting DNA - restriction enzyme
Joining DNA - T4 ligase
Explain how recombinant DNA and GMOs are made, and especially
which enzymes do which jobs in this process
Digesti
on and
ligation
Some
plasmid
s that
are
recomb
inant
(contai
n bits
of
foreign
DNA)
Starting
plasmid
(non-
recomb
In ligation
mixture…
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Document Summary

Early fermentations used mixed cultures of naturally occurring bacteria and fungi e. g. scandinavian fermented fish storage unit c. 7000 bc. Earliest firm evidence for alcoholic fermentation around 7000 bc in china, analysis of jars revealed traces of alcohol and plants used for fermentation. Relationship with fermenting yeasts is ancient, and remains very strong. Explain why microbes are useful for biotechnology, using examples of specific fungi (s. cerevisiae) and bacteria (e. coli) Archaea source of thermostable polymerase enzymes for copying dna sequences. Bacteria excellent hosts for cloning dna and expressing proteins. Conversion of co2+ light into biofuels (ethanol, h2) Contains machinery for biosynthesis of high-value products form simple raw materials. Very easy to extract or add plasmid dna. Generally recognised as safe (gras), can"t cause infection. Explain what a plasmid is, and define the roles of different kinds of plasmids in nature and in biotechnology. Host cell factory needs instructions (ie. dna) or blueprint to tell it which products to make.

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