Different mutations are created in the commercial host strains of E.coli which provide them with advantages as compared to the wild type strain. This presentation deals with few strains like Dh5alpha, DH10B, Bl21, JM109 E.coli strains and their associated mutations.

1. 1

2.  Commercial hosts for transformation and cloning have been created by making multiple gene
mutations in the genome of the organism
 Protein expressed from high copy number plasmids and from powerful promoters can use up the
host machinery and can slow the growth of the host.
 Some protein products may be lethal to the host.
 Certain specialized strains are now available commercially to impart greater transcriptional control,
assist with protein folding, and reduce codon bias.
2

3. Use of modified strains
Fast growth
Protein
stability
Routine
cloning
Cloning of rare
sequences
■ Most of the commercial strains are marketed for different purposes
Cloning of
unstable DNA
Preparing
unmethylated
DNA
High throughput
cloning
3

4. E.coli strains – wild type
■ The majority of all common, commercial lab strains of E.coli used today come from two strains of
E.coli: the K-12 strain and the B strain.
■ The K-12 was isolated from the faeces of a diphtheria patient in 1922 and eventually led to the
common lab strains MG1655 and its derivatives DH1.
■ DH1 led to production of DH5α and DH10β (also called TOP10).
■ K12 also gave rise to strains like W1485 and W2637.
■ The B strain of E. coli was isolated in 1918 and gave rise to BL21 strain and derivatives.
4

5. DH5α E.coli cells
■ Widely used E.coli strain to clone cells and to maximize transformation efficiency.
■ The name come from Douglas Hanahan.
■ The cells have been mutated majorly in genes:
– recA1: mutation in the DNA-dependent ATPase that is essential for recombination and DNA
repair. This reduces plasmid recombination and increases plasmid stability.
– endA1: mutation in the endonuclease I enzyme. Prevents the endonuclease from cleaving the
incoming plasmid DNA and improves the stability of plasmid in the host.
– Δ(lacZ)M15: expresses the β fragment of beta galactosidase.
– hsdR17: inactivates the host restriction-modification system. Incoming DNA gets methylated, but
not restricted.
5

6. RecA
■ RecA is an ATP-dependent DNA repair protein that
catalyzes the DNA strand exchange reaction in
homologous recombination
■ RecA along with a recBCD enzyme constitute the
RecBCD pathway of homologous recombination
■ RecA monomers polymerize onto DNA in the
presence of ATP to form a nucleoprotein filament
that is competent to promote DNA strand exchange
■ Mutation at specific residues - Lys216 , Phe217 or
Arg222 – make the enzyme unable to polymerise
onto ssDNA
6

7. EndA mutation
■ Endonuclease I is a 12kDa periplasmic protein encoded by the gene endA that degrades double-
stranded DNA.
■ The E. coli genotype endA1 refers to a mutation in the wildtype endA gene, which produces an
inactive form of the nuclease.
■ endA gene mutation results in the reduction of endogenous levels of nuclease activity and increases
the yield and quality of purified plasmid molecules.
■ Strains carrying the endA1 mutation produces higher quality plasmid DNA preparations.
7

8. LacZ∆M15
■ The lacZΔM15 mutation deactivates lacZ activity in the
bacteria producing an inactive form of β-galactosidase.
■ Strains with this mutation can not cleave X-gal and
remain colourless on X-gal plates.
■ If a plasmid carrying a lacZ alpha subunit is introduced
into the strain, it complements the truncated lacZ gene
and produces β- galactosidase activity.
■ An insert at the MCS of such plasmids will disrupt the
alpha subunit and colonies containing an insert will
appear as white colonies rather than blue ones. (Blue
White screening)
8

9. hsdR17
■ Many E.coli strains contain restriction-modification system encoded by the hsdRMS locus.
■ The hsdR gene encodes an endonuclease that cleaves DNA containing the sequence -
AACNNNNNNGTGC-, unless the 2nd of the two A residues and the adenine residue on the
other strand opposite the thymine are methylated.
■ An hsdR mutation avoid cleavage of incoming unprotected DNA.
■ The hsdM gene encodes the methylase that protects against degradation
■ Cloning DNA in an hsdR-M+ strain can be used to allow methylation if it is subsequently
necessary to propagate in an hsdR+ strain
9

10. Other mutation in DH5α
■ thi-1: This strain requires thiamine (thiamine auxotroph)
■ relA1: Increases strength of the bacterial cell membrane and also allows RNA synthesis in the
absence of protein synthesis.
■ nupG: Encodes broad-specificity transporter of purine and pyrimidine nucleosides. Mutation allows
constitutive expression of deoxyribose synthesis genes and permits uptake of large plasmids.
■ deoR: Regulatory gene mutation allowing constitutive expression of genes for deoxyribose
synthesis. Allows efficient propagation of large plasmids.
10

11. DH10B E.coli cells
■ DH10B competent E. coli cells provide high transformation efficiency which makes them ideal for
generating cDNA or genomic libraries.
■ The major mutations seen includes the elimination of mcrA, mcrBC, mrr, and hsdRMS restriction
systems to allow construction of more representative genomic libraries.
■ Also possess recA, endA and lacZΔM15 mutations
■ A variant of the DH10B E.coli host also possess a mutation in the tonA gene, which confers resistance
to bacteriophages like T1, φ80 and T5 phages.
11

12. mcrA, mcrB and mrr
■ There are proteins in E.coli strain K that will degrade incoming DNA if it is methylated, belonging
to the methylation dependent restriction systems (MDRS).
■ Products of the mcrA, mcrB and mrr loci are endonucleases, which will degrade DNA containing
methylcytosine (mcrA, mcrB) or methyladenine (mrr).
■ Using strains mutant in these loci, therefore, is desirable, particularly when cloning highly
methylated DNA.
12

13. BL21 and BL21(DE3) E.coli cells
■ E.coli BL21 and BL21(DE3), created by F. William Studier and Barbara A. Moffatt, are common
laboratory strains for recombinant protein production.
■ The lack of lon and ompT proteases, often regarded as common characteristics among B
lineage. Increases the stability of the protein product.
■ Mutation of dcm – methylation of cytosine inhibited.
■ E.coli BL21(DE3) harbors a prophage DE3 derived from a bacteriophage λ, which carries the T7
RNA polymerase gene under the control of the lacUV5 promoter.
13

14. lon and ompT proteases
■ Lon is an ATP-dependent cytoplasmic protease which degrades misfolded proteins and certain
rapidly-degraded regulatory proteins.
■ OmpT is an aspartyl protease found on the outer membrane (periplasm) of E.coli, which was found
to cleave antimicrobial peptides, certain proteins and degrade some recombinant heterologous
proteins.
■ The use of host strains carrying mutations which eliminate the production of proteases can
enhance accumulation of protein of interest by reducing proteolytic degradation.
14

15. dam and dcm
■ Methylation can be carried out by the products of the dam and dcm genes.
■ The Dam protein methylates adenines at the sequence -GATC-
■ Dcm methylates cytosines at the sequences -CCAGG- and -CCTGG-.
■ Some frequently used restriction enzymes have recognition sites that overlap with these and are
inhibited by methylation, so DNA prepared from strains that are wild type for these loci will not be
efficiently restricted by restriction enzymes.
■ Dam and dcm mutations useful for preparing unmethylated DNA, important when trying to cut with
certain restriction enzymes (ex: ClaI or XbaI)
15

16. JM109 E.coli cells
■ JM109 competent E. coli contains mutations in recA1 and endA1 genes. These mutations aid in
minimizing recombination and ensuring plasmid stability.
■ The F′ factor is suitable for the growth of bacteriophages (such as M13) to obtain single stranded
DNA.
■ thi-1: Thiamine auxotroph
■ mcrB+: McrB system of E. coli interferes with incoming DNA containing methylcytosine. Could
interfere with cloning experiments
■ lacZΔM15: This E. coli strain carries the lacZ deletion mutant which contains the ω-peptide: a
mutant β-galactosidase derived from the M15 strain of E. coli that has its N-terminal residues
11—41 deleted and is unable to form a tetramer so it is inactive.
16

17. ■ dut: dUTPase, an enzyme that prevents the incorporation of uracil into DNA by destroying dUTP.
Dut-ung double mutants accumulate a significant amount of uracil in their DNA.
■ Ung: Mutant in uracil N-glycosylase, an enzyme that removes uracil from DNA. An ung mutant
allows uracil to persist in DNA
■ e14: A prophage-like element, present in K-12 but missing from many derivatives. e14 carries the
mcrA gene, so e14- strains are McrA-.
■ F: A self-transmissible plasmid that confers the ability to make pili and thus to be infected by
male-specific phage like M13.
■ lacIQ: Overproduces the lacI gene product, a repressor of the lac operon.
■ Δlac: There are three common deletions involving the entire lacZYA operon in addition to some
flanking DNA: ΔU169, Δ X111, and ΔX74.
■ recB, recC, recD: Required for ExoV function. Recombination deficient.
■ recF, recJ: Recombination gene required for interplasmid homologous recombination.
17

18. ■ sbcB: Required for Exonuclease I function. Strains carrying recB recC and sbcB are usually also
sbcC. These quadruple mutant strains are recombination-proficient and propagate inverted repeats
in λ, but plasmid replication is aberrant.
■ sbcC: Helps (with sbcB) to supress the effect of a recBC mutant. sbcC mutants are Rec+ and
stably propagate inverted repeats in plasmids.
■ supE: Mutant tRNA inserts glutamine at UAG codons, suppressing UAG mutations in the reading
frame. SupE is required for the lytic growth of some phage mutants.
■ supF: (or tyrT) Mutant tRNA inserts tyrosine at UAG codons, thus suppressing the effect of UAG
mutations in reading frames. This mutation is required for the lytic growth of some λ phage, such
as λgt11.
■ φ80dlacΔM15: Lysogenic for a defective prophage derivative of φ80. The lacZ ΔM15 allele
supplies the beta peptide for blue white screening.
18

19. 19