Jess Oscar Lacayo Gutirrez A01327469

Ingeniera
Gentica BT2001.1
Dra. Hera Andrade Z.
2.2 E. coli strains for
genetic engineering uses
1.- What is the strain, from wich most of the GE used strains are derived?
The strain is important for recombinat DNA experiments. Almost all
strains currently used in recombinant DNA experiments are derived form
a single strain: E. coli K-12, isolated form the feces of a diphtheria
patient in 1922.
2.- Describe in detail the system to indicate the E. coli genotypes
In an organism, the genetic state of their DNA can be indicated by their
genotype. It is associated by its counterpart, the phenotype, an observer
behavior, that is visual for the human eye or is most noticed than the
genotype. Genotypes of E. coli strains are described with a standard
nomenclature proposed by Demerec. Genes are given three-letter,
lowercase, italicized names that are often mnemonics suggesting the
function of the gene.
3.- Mention at least 5 examples of E. coli genotypes and their meaning
Properties of Common Genotypes of E. coli Host Strains
Mutation
Amy

Description
Expresses amylase

Significance
Allows

ara

utilization
Mutation in arabinose Blocks

dam

metabolism
Blocks

amylose
arabinose

utilization
adenine Makes

DNA

methylation at GATC susceptible

sequences
dcm

Blocks
methylation

cleavage

by

some

restriction enzymes
cytosine Makes
DNA
at susceptible

CC(A/T)GG sequences
(DE3)

to

cleavage

to
by

some

restriction enzymes
lysogen carrying Used for T7 promoter-

Jess Oscar Lacayo Gutirrez A01327469

Ingeniera
Gentica BT2001.1
Dra. Hera Andrade Z.
2.2 E. coli strains for
genetic engineering uses
the gene for T7 RNA based
expression
polymerase

systems

4.- What are amber and ochre mutations?

They are nonsense mutations. An amber mutation is a nonsense
mutation that changes a sense codon (one specifying an amino acid)
into the translational stop codon UAG, causing premature termination of
the polypeptide chain during translation. An ochre mutation is a
nonsense mutation that changes a sense codon (one specifying an
amino acid) into the stop codon UAA, causing premature termination of
the polypeptide chain during translation.
5.- Explain the genotypes of the following E. coli strains: DH5 , XL1blue, SURE, MRF
DH5 :deoRendA1gyrA96hsdR17(lac)U169recA1relA1supE44thi1(80lacZ
M15)
Contains the phi-80deltalacZDeltaM15 marker which provides alphacomplementation of the beta-galactosidase gene allowing for blue/white
screening. Carries recA1 for insert stability of recombinants and endA1
for improving the quality of plasmid DNA. Restriction negative (hsdR17).
For generating cDNA libraries and subcloning.
XL1-blue: endA1 gyrA96 lac

(mcrA)183

(mcrCB-hsdSMR-

mrr)173 recA1 relA1

Nalidixic acid resistant tetracycline resistant (carried on the F plasmid)
SURE: endA1 gyrA96 lac mcrA

(mcrCB-hsdSMR-mrr)171 recB recJ

relA1sbcC supE44 thi-1 umuC::Tn5(Kanr) uvrC F'[lacIq lacZ M15 proAB+]

Jess Oscar Lacayo Gutirrez A01327469

Ingeniera
Gentica BT2001.1
Dra. Hera Andrade Z.
2.2 E. coli strains for
genetic engineering uses
Uncertain status of TraD36 in F plasmid. Increased stability for inverted
repeats

and

Z-DNA.

Recombination-deficient.

Relaxed

phenotype.

Endonuclease A-deficient. Constitutive lac repressor expression. Amber

suppressor UAG>CAG(Gln). Improved cloning 5-mC DNA. Nalidixic acid
resistant. Kanamycin resistant. Tetracycline resistant.
MRF: supE44thi1F'[lacIqlacZM15proAB+Tn10(Tetr)AmyCamr]
Used in lambda phage infection, amplification, expression. Tetracyline
resistant. Stratagene E. coli Genotype Strains

6.- What is the F factor?

Some E. coli strains carry an F episome or fertility factor, which can be
found

in

several

different

forms.

In

Hfr

cells

(high-frequency

chromosome donation), the F factor is integrated into the bacterial

chromosome and can cause chromosomal transfer. Strains containing
the F factor produce surface pili, which are required for infection by
vectors based on filamentous phage.
7.- What are the restriction and modification systems?
Restrictionmodification systems have the function of prevent the
genetic exchange between different groups of bacteria, they make
that by enabling the host to recognize and destroy the foreign
DNA.
8.- Explain in a graphic form the Dam and Dcm Methylation, the EcoK
System and the McrA, McrBC, and Mrr Restriction systems.
9.-What is recombination and how it works for E. coli strains
AfterasuccessfulrecombinationofaplasmidvectorintoE.coli,thehostrecombinationsystemcan
catalyzetherearrangementoftherecombinantmoleculeofE.coli,andwiththisspecificproblemthe
DNAcanduplicatebecausecancontaindirectandinvertedrepeats.

10.-Mention host for mutagenesis

Jess Oscar Lacayo Gutirrez A01327469

Ingeniera
Gentica BT2001.1
Dra. Hera Andrade Z.
2.2 E. coli strains for
genetic engineering uses
Its possible the increase of a spontaneous mutation in E. coli by
nearly three or four orders of magnitude by mutation in mutD gene
which encodes in 3 to 5 exonuclease sub unit of the DNA
polymerase III, the site directed mutagenesis methods involve
certain intermediates to contain wild type/mutants.
11,- Mention 3 specialized strains for recombinant protein production
examples and their genotypes meaning
BL21df: al hsdSB ompT, protease deficient
BL21,trxBd-e ,gal hsdSB ompT trxB15::Kanr, Enhances cytoplasmic
disulfide N bond formation
B834d,e gal hsdSB met ompT, Protease deficient; used for N
labeling with 35S-methionine
12.-Explain the main features for recombinant protein expression
Repressors, the E. coli expression vectors use active induced
promoters and with that correct the host strain to ensure the tight
regulation , stability, sometimes the host proteases could interfere
with the isolation of recombinant proteins, the purpose is to avoid
the degradation by the use of protease deficient hosts , codon
bias, can have an impact in the heterologous protein expression,
so the genes with a very high proportion of rare codons are poorly
expressed , solubility and posttranslational processing , because if
the heterologous proteins are overprotected can result in
misfolding and segregation into a insoluble inclusion bodies, the
DnaK-DnaJ and GroES-GroEL assist in folding wild type E. coli and
there is evidence that overproduction of that complex increase the
yield of soluble proteins form recombinant E. coli.