E. coli

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Dec 14, 2012 (4 years and 8 months ago)

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Biotech Certification 06 Fall

1

Lecture 2

Plasmid Biology

Genetic Transformation

Biotech Certification 06 Fall

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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.

Biotech Certification 06 Fall

3

The Replication of Plasmid


A replicon
-

self
-
replicating genetic unit.


Either replicate as host cell divides or will be lost.


Bacteria doesn’t ‘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 other’s replication.

Biotech Certification 06 Fall

4

Plasmid Applications


Works as a genetic shutter to deliver genetic
material.


Create genetic library for an organism.


Facilitate the Sequencing an organism’s 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.




Biotech Certification 06 Fall

5

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




polylinker

Biotech Certification 06 Fall

6

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 P
BAD

and P
CMV
Promoters).


PloyA terminal signal



terminate the transcription of
cloned gene.


Ori site



to initiate the replication of the plasmid.



Biotech Certification 06 Fall

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Plasmid used in our class

polylinker

PBAD

Selection Marker
:

Lactamase gene
-

ampicillin resistance.

E.Coli take up this plasmid can grow LB

medium with antibiotics ampicillin.

Promoter
:

P
BAD

inducible promoter
-

Under control

of araC regulatory protein.

How does this work ?

Under normal condition, araC Protein

generally was bond to P
BAD
. So, the gene downstream of P
BAD

won’t

be expressed.

However, araC Protein will release from the promoter if the bacterial

Bearing this plasmid grow in LB medium with arabinose (
Inducer
).

Biotech Certification 06 Fall

8

Genetic transformation


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


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


Biotech Certification 06 Fall

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Bacteria Transformation with Plasmid



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
0
C (
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.


There are two methods to transform
E. coli

cells with

Plasmids.


Biotech Certification 06 Fall

10

Transformed Bacteria E.Coli Culture


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



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

Biotech Certification 06 Fall

11

Outline genetic engineering

polylinker

+

Jelly fish
genome

The whole Genome

Fragments


Jelly Fish Genome

Library

ligation

Transformation

fragmentation

selection

Biotech Certification 06 Fall

12

Today’s 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.