Let’s agree some SUBSTANTIAL RULES

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Genotype Nomenclature
I. Genotypes of E. coli strains are described in
accordance with a standard nomenclature
proposed by Demerecet( al ., 1966)
II. By convention, E. coli genotypes list only
defective genes, but the superscript symbols
“–” and “+” are used to emphasize a wild-type
locus
III. Genes are given three-letter, lowercase,
italicized names that are often mnemonics
suggesting the function of the gene
IV. If the same function is affected by several
genes;
the different genes are distinguished with
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uppercase italic letters, for example rec A,
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Genotype t
Nomenclature
e.g. rpsL
o(Str r
A mutation
) in the gene for ribosomal protein
small subunit S12
confers resistance to
V. Phenotypes are capitalized and the letters are
streptomycin
followed by either superscript “+” or “–” or
sometimes “r” for resistant or “s” for sensitive.
VI. Specific mutations are given allele numbers
that are usually italic arabic numerals such as
hsdR17 .
VII. A constitutive mutation is denoted by
superscript q; thus lacIq indicates constitutive
expression of the gene for the lac repressor
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Genotype Nomenclature
VIII. Deletions are denoted by △. If △ is followed by
the names of deleted genes in parentheses, as
in △(lac-pro), then all of the genes between the
named genes are also deleted.

IX. An insertion is indicated by “::” preceded by

the position of the insertion and followed by
the inserted DNA; for example,trpC 22::Tn10
trpC .
denotes an insertion of Tn10 into

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o Various Escherichiat coli host strains are used
for the propagation and manipulation of
recombinant DNA

o E.coli host should contain certain mutations

that are relevant to recombinant DNA
experiments

o Almost all strains used in recombinant DNA

experiments are derived from a single strain:E.
coli K-12

o E. coli K-12 unlike wild-type strains

well-adapted to the laboratory environment
have lost their ability to thrive in the intestine.
lose their ability to form biofilms 5
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o Derivatives of E. t K-12
coli normally contain
three site-specific DNA methylases: Dam, Dcm
and EcoK.
I. DNA adenine methylase, encoded by dam ,
methylates adenine residues in the sequence
GATC
Ȳ This sequence will occur approximately once
every 256 bp in a theoretical piece of DNA of
random sequence

II. DNA cytosine methylase, encoded by dcm ,

methylates the internal cytosine residue in the
sequence CC(A/T)GG
Ȳ This sequence occurs on average once every 512
bp 6
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o All commonly t cloning
used strains are Dam+
Dcm+
o Strains that are recA – are always dam +, because
the combinationrecA – dam – results in a lethal
phenotype

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I. In order to:
✔ check whether a successful recombinant
plasmid has been constructed
✔ obtain multiple copies of target gene
transformation step must be executed in certain
strain ofE. coli known as cloning strain (DH5 α)

II. In order to:

✔ express target gene
transformation step carried out in E. coli
expression strain (BL21)

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Cloning
t
DH5
Strain: α

I.
In order to protect plasmid DNA from inner-
nucleases action
II
In order to suppress new recombination between plasmid
.and bacterial chromosome

III
.
IV
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Expression t
BL
Strain: 21
BL
21

I.

II
.

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The pET vector is a little different from the
pUC vector:
o pUC uses thelac promoter and pET uses
a promoter from phage T7
o The phage T7 promoter is stronger than
thelac promoter
o Phage T7 RNA polymerase will
specifically recognize the T7 promoter
region and will not efficiently transcribe
from other promoters
o The will not be efficiently transcribed
byE. coli RNA polymerase 12
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o The pET systemt involves not only an
expression vector, but also a genetically
engineered host bacteria.
o The host bacteria for the pET vector is
typicallyE. coli strainBL(DE3)
o This strain has integrated into its chromosome
the gene for T7 RNA polymerase
o The T7 RNA polymerase in the host genome is
constructed such that it is under the control of
lac promoter and operator
a
o Thus, induction by the lactose analogue, IPTG,
causes the host to produce T7 RNA polymerase
o TheE. coli host genome also carries
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thelacI (repressor) gene
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o Target genes are cloned under strong T7
bacteriophage promoter.

o The expression of the target protein is

inducible by providing T7 RNA polymerase in
the host cell as an inducing signal.
o Target gene is initially cloned to host cell that
do not contain T7 RNA Polymerase, thus
increasing plasmid stability.

o Once stabilized in a non-expression host, the

recombinant plasmid is transferred to a
expression host having T7 RNA Polymerase
gene in the genome.
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