Plasmid Biology

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Lecture 2

• Plasmid Biology
• Genetic Transformation

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What is plasmid?

• An autonomously replicating mini-chromosomes.
• It was found in the bacteria.
• Double strand DNA molecule.
• Most of them are circular, linear plasmids have
  also been identified.
• Some can freely transfer between bacteria.

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The Replication of Plasmid

• A replicon - self-replicating genetic unit.
• Either replicate as host cell divides or will be
  lost.
• Bacteria doesnt spit out plasmid DNA.
• Two functions of replication (replication and
  partitioning).
• Replication - cause high copy plasmids.
• Partition each progeny cell receives a copy of
  plasmid (low copy plasmids).
• Replication Incompatible two different plasmids
  will interfere with each others replication.

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Plasmid Applications

• Works as a genetic shutter to deliver genetic
  material.
• Create genetic library for an organism.
• Facilitate the Sequencing an organisms genome.
• Express a protein by cloning a gene to its
  backbone.
• Express a RNA molecule.
• Hold a piece of DNA to use as hybridization
  probe.

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Genetically Modified Plasmid

• The plasmids used in biotech field are
  genetically modified plasmids.
• A typical genetically modified plasmid has
  following components
• - Origin of replication
• - Promoter
• - Polylinker
• - PolyA signal tail sequence
• - Selection marker

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Function of these components

• Selection marker- used to isolate host cells
  taken up your plasmid (e.g. lactamase and lacZ
  genes.).
• Polylinker (multiple cloning site) - used to
  clone DNA fragments to the backbone of plasmid.
• Inducible or uneducable Promoter used to
  control the expression of the cloned genes (e.g
  PBAD and PCMV Promoters).
• PloyA terminal signal terminate the
  transcription of cloned gene.
• Ori site to initiate the replication of the
  plasmid.

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Plasmid used in our class
Selection Marker Lactamase gene- ampicillin
resistance. E.Coli take up this plasmid can grow
LB medium with antibiotics ampicillin. Promoter
PBAD inducible promoter- Under control of araC
regulatory protein. How does this work ? Under
normal condition, araC Protein generally was bond
to PBAD. So, the gene downstream of PBAD wont be
expressed. However, araC Protein will release
from the promoter if the bacterial Bearing this
plasmid grow in LB medium with arabinose
(Inducer).
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Genetic transformation

• Genetic transformation is the way by which an
  organism acquires and expresses a new gene.

• Genetic engineering directed transfer of a gene,
  or piece of DNA into a cell (typically a
  bacteria), simply speaking, to force the cell to
  take up a piece of DNA.
• The purpose of forcing cell to take up DNA is to
  force the cell to express (produce) the protein
  that the DNA code.
• Materials used to performing transformation (DNA
  plasmid, Cell E.coli Bacteria

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Bacteria Transformation with Plasmid

• There are two methods to transform E. coli cells
  with
• Plasmids.

• Chemical transformation- cells are grown to
  mid-log phase, harvested and treated with CaCl2.
  Cells treated in such a way are said to be
  competent. The competent cells are mixed with the
  DNA on ice, followed by a brief incubation at 42
  0C ( heat shock).
• Electroporation transformation- cells are also
  grown to mid-log phase but are then washed
  extensively with water to eliminate all salts.
  washed E. coli are mixed with the DNA to be
  transformed and then pipetted into a plastic
  cuvette containing electrodes. A short electric
  pulse, about 2400 volts/cm, is applied to the
  cells causing smalls holes in the membrane
  through which the DNA enters.

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Transformed Bacteria E.Coli Culture
E. Coli can be cultured in LB medium( providing
enough and necessary nutrition for bacteria
growing).

• There are two kinds of LB media (liquid and solid
  media).
• Liquid medium used to propagate bacterial cells.
• Solid medium used to isolation and propagation of
  E. coli colonies.
• Upon transformation, the E.coli cells should grow
  in LB liquid medium to recover the transformed
  cells for 45-60 min.
• And then plated on solid LB agar medium, usually
  prepared with selection reagents

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Outline genetic engineering
Jelly fish genome
fragmentation

polylinker
ligation
The whole Genome Fragments
Jelly Fish Genome Library
Transformation
selection
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Todays Experiment

• You will perform transformation E.coli cells with
  Jelly fish genomic library constructed with
  plasmid vector.
• You will perform plating transformed E.coli cells
  on selective LB agar plates.
• You will grow your cells by incubating them in an
  optimal condition.